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Fluoride Ion Sensing in Aqueous Medium by Employing Nitrobenzoxadiazole-Postgrafted Mesoporous Silica Nano Particles (MCM-41) Gaurav Jhaa, Anoop N. b, Abdur Rahamanb and Moloy Sarkar*a 5
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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x Mesoporous Silica-based inorganic-organic hybrid material (NBD-AP-MCM) as fluorescent chemosensor for detection of fluoride in aqueous medium has been designed and developed. The system has been developed by covalently anchoring 7-nitro-2, 1, 3-benzoxadiazole (NBD) dye to the surface of mesoporous silica nano particles, MCM-41. The system has been characterized by several conventional analytical methods comprising spectroscopic, microscopic and thermo-gravimetric techniques. Sensory action of the material has been investigated by carrying out steady state absorption and fluorescence and time resolved fluorescence studies on the system in absence and presence of several biologically and environmentally important anions in aqueous solution. Photophysical data of the present system (NBDAP-MCM) are also compared with the free dye (NBD) molecule. Significant decrease in the fluorescence quantum yield of the fluorophore in the hybrid material NBD-AP-MCM has been observed as compared to the unbound NBD. The decrease in fluorescence efficiency in hybrid material is attributed to the aggregation caused quenching (ACQ) phenomenon. Interestingly, the system displays more than six fold fluorescence enhancement in presence of fluoride ion in aqueous solution. Enhancement of fluorescence lifetime of the fluorescing moiety (NBD) has also been observed during fluorescence time-resolved studies. No significant optical changes have been observed with other commonly encountered anions rendering the present system highly selective towards fluoride detection. The fluorescence enhancement has been attributed to the cleavage of Si-O bonds due to the addition of fluoride. The silyl cleavage detaches the fluorophore from the solid support and thereby making the fluorophore “free” in solution which in turn recovers its original fluorescence which was decreased because of the aggregation on solid silica support. Furthermore, the suitability of the present system in cellular imaging has also been demonstrated.
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Fluoride is an essential anion, abundantly present in soil, ocean and ground water1. Its utility in prevention of dental caries, and treatment of osteoporosis, has been explored widely2. Fluoride ions are also detected as an indicator of uranium refinement (via hydrolysis of UF 6) or of the chemical warfare agent sarin, which releases fluoride ion upon hydrolysis. 2 However, overexposure to fluoride is harmful for biological systems and in human beings, it causes nausea, fluorosis, abdominal pain, coma, cardiac arrest etc.2,3 In the light of these problems, development of fluoride detection techniques have gained widespread attention in recent years, especially using fluorescence technique, which allows high sensitivity, low detection limit and is capable of intracellular fluoride detection.2-4 The chemosensors reported in literature, often utilize fluoride promoted bond cleavage,4-7 boron−fluoride interaction3, This journal is © The Royal Society of Chemistry [year]
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hydrogen-bond and π−π interaction8,9, and metal binding10,11. But majority of the chemosensors reported, require organic medium, since aqueous medium poses problems regarding solubility, polarity and hydrogen bonding. 12,13 Moreover, one inherent problem for detection of fluoride ion in aqueous medium is due its high hydration energy. 14 It may be mentioned that sensors discriminating through fluorescence quenching are prone to lower selectivity of sensing as compared to those based on fluorescence enhancement. Very few probes have been reported in literature, which actively sense fluoride without these restrictions, 6,15,16 for practical application of fluoride detection in aqueous solution. Moreover, most sensors are still too large for a number of conceivable applications and miniaturization to the nanoscale is essential for widespread applicability. Hence, development of adequate nanomaterials, is an important direction for sensor technologies. One of the interesting physical attributes of nano
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Scheme 1. Schematic representation of postgrafting of 7-nitrobenz-2oxa-1,3-diazol-4-yl (NBD) probe on MCM-41 surface. 5
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particles is their ability to provide a much larger surface to volume ratio compared to that of bulk material. Decreasing the size of the sensor has several advantages, which include the need for assaying smaller sample volumes, lowering available detection limits and reducing the amount of the involved sensing chemicals. In view of this, it is highly important to develop new sensing methodologies by exploiting nanoparticle –based sensor systems towards a simple, inexpensive, sensitive and selective detection of fluoride ion in aqueous medium. Mesoporous silica materials (e.g. MCM-41) are emerging as a new versatile, non-toxic and water dispersible solid support for immobilization of fluorogenic probes on their surface for optical sensing device fabrication17,18. The characteristic properties of mesoporous silica materials, such as high surface area which allows large probe doping concentrations, and highly uniform porosity enabling facile diffusion of analyte molecules, renders these materials to be an excellent support for photoluminescent sensing probes19,20. Such mesoporous material-fluorophore conjugates are widely used for small molecule and ion sensing, drug-delivery and in-vivo cellular imaging purposes. 21 Compared to free dye molecules, which are prone to photobleaching, solvatochromic effects and often display cellular toxicity, mesoporous silica based fluorescent probes are highly biocompatible, demonstrate enhanced per particle brightness and there surface can be easily functionalized for biological targeting by anchoring proteins, antibodies or cell penetrating peptides. 22-24 The optical transparency in UV and visible range, 18,20 of these materials, enables the exploration of probe behaviour on solid support as well as their transition from solid to solution phase. 2 | Journal Name, [year], [vol], 00–00
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There is an additional requirement of an environment sensitive probe, to signal the event of this phase transition from solid to solution, upon the attack of an analyte moiety detaching the probe from the solid support. Such a system can selectively discriminate between analytes, based on specific reactions, and give rise to a highly selective optical sensor that is capable of detecting analytes in aqueous medium at low detection limit. In the present work, we have devised a new fluoride anion sensor, consisting of a 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) probe, covalently anchored to MCM-41 surface. NBD probe has been covalently grafted on MCM-41 surface by means of 3(aminopropyl)triethoxysilane, in order to prevent the leaching of dye molecules, in solution phase17. NBD is chosen as the signalling fluorophore for this system, due to its charge transfer based highly robust fluorescence, environment sensitivity and the ease of reaction pertaining to probe labelling, which has been extensively exploited, both in vitro and in vivo applications. 25 The NBD moiety is in quenched state when attached to the MCM-41 surface in acetonitrile/water (7:3) mixture. Fluoride ion exhibits a nucleophilic attack on the Si-O bond, which is responsible for holding together the silica surface and the fluorophore, thereby releasing the NBD moiety into the aqueous/organic solvent system, where the probe, regains its fluorescence and signals the presence of fluoride through an enhancement in fluorescence. This transition of NBD from solid support to solution has been monitored via steady state and time dependent fluorescence studies. The affinity of fluoride ion for Si-O and Si-C bonds has been extensively studied and utilized for design of fluoride ion sensing probes where fluoride attack leads to generation of more fluorescent chemical species, 2,4-7 mostly anions, but these probes require the resulting anions to be in conjugation with the fluorogenic system. Designing such conjugated systems require extensive organic synthesis and tricky purification steps. However, the sensor design using postgrafting of mesoporous silica requires nominal synthesis and easy purification, with considerably higher yields. Furthermore, this work has been inspired form the considerable deficit of studies in the literature, focusing on the detailed photophysical analysis of the behaviour of ion sensing probes, postgrafted on the solid surface of mesoporous silica particles. These studies are important to provide better understanding of the mechanisms of ion sensing and dynamics of the probe at the silica surface, and to further devise probes having better sensitivity, selectivity and simpler construction 20.
2. Material and Methods 80
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The absorption and emission spectra are recorded on a UVvisible spectrometer (Cary100, Varian) and a spectrofluorimeter (Perkin Elmer, LS 55), respectively. The fluorescence spectra are corrected for instrumental response. Time resolved fluorescence measurements are carried out by a time-correlated single photon counting (TCSPC) spectrometer (Edinburg, OB920). Picosecond pulse diode laser (EPL-405) is used as excitation source, and an MCP photomultiplier (Hamamatsu R3809U-50) is used as the detector (response time 40ps). The instrumental response time of our instrument is about 98 ps (FWHM). The lamp profile is recorded by scaterer (dilute ludox solution in water) in place of This journal is © The Royal Society of Chemistry [year]
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the sample. Elemental analysis through EDX (energy-dispersive X-ray spectroscopy) and scanning electron microscopy (SEM) images of postgrafted mesoporous silica were obtained using field-emission scanning electron microscope (FESEM) system (Carl Zeiss, Germany make, model: Pigma). The SEM samples were prepared by drop casting dispersion of postgrafted mesoporous silica in acetonitrile: water (7:3), and evaporating in air at room temperature. Powder X-ray diffraction (p-XRD) patterns were performed on a Bruker DAVINCI D8 ADVANCE diffractometer equipped with Cu Ka radiation (l ¼ 0.15406 nm). FT-IR measurements of the system were performed using a Perkin-Elmer Spectrum RXI FT-IR spectrophotometer, and thermogravimetric analysis (TGA) was carried out using PerkinElmer Simultaneous Thermal Analyzer STA-6000. Fluorescence microscopy images were taken in Axio Imager.M2 at 40X magnification, detected with filter AF 488. Mesostructured silica, MCM-41 type, 4-chloro-7nitrobenzofurazan (98%), sodium azide (>99.5%), tetrabutylammonium fluoride hydrate (98%), tetrabutylammonium chloride (>97.0%), tetrabutylammonium bromide (>98%), tetrabutylammonium iodide (purum >98%), tetrabutylammonium acetate (>97%) and tetrabutylammonium bisulphate(purum, >97%) were obtained from Sigma-Aldrich and were used without any further purification. 3-aminopropyl triethoxysilane(APTES) was obtained from Himedia and was used without any further purification. Acetonitrile used in photophysical studies was anhydrous and was purchased from Sigma-Aldrich.
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Fig 1. FE-SEM images of NBD-AP-MCM (upper panel) scale bar = 200 nm, EDX micrograph (lower panel, left side) and elemental composition of NBD-AP-MCM (lower panel, right side). ). Elemental Composition : Carbon (C)=19.9%, Nitrogen (N)=5.0%, Oxygen(O)= 37.7%, Silicon(Si) = 37.7%.
3. Experimental Section 65
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a) Synthesis of NBDH: NBDH (7-nitrobenzo[c][1,2,5]oxadiazol4-amine) was prepared from 4-chloro-7-nitrobenz-2-oxa-1,3diazole (NBD-Cl) by following a standard procedure. 26 The compound was characterized by 1H NMR. Yield of NBDH ~ 50%. 1H NMR of NBDH (DMSO-d6, ppm): δ 8.89 (s, 2H), 8.52 (d, 1H), 6.42 (d, 1H). b) Synthesis of AP-MCM: APTES (3-aminopropyl triethoxysilane) was anchored to MCM-41, using a reported procedure 27. 500 mg of MCM-41 was pre-treated at 180 °C for 2 h and dispersed in 30 mL of dry toluene. After the addition of APTES (160 μL for the targeted functionalization degree of 1.2 mmol per gram of parent MCM-41), the suspension was refluxed for 3 h. The functionalized product was recovered by filtration, washed with 100 mL of ethanol, and oven-dried at 80 °C for 1 h. c) Synthesis of NBD-AP-MCM: 100 mg of dried AP-MCM was dispersed in 10ml ethanol. 100 mg (0.5 mmol) of 4-chloro-7nitrobenzo[c][1,2,5]oxadiazole (NBD-chloride) was added to the solution and the resulting mixture was left stirring for 2-3 hours. The functionalized product was recovered by filtration, washed with ethanol, dichloromethane and water and oven-dried at 110 °C for 2 hours. d) Sample Preparation for Anion Sensing: A general procedure was followed to study the interaction of anions, with NBD-APMCM. The synthesized NBD-AP-MCM was suspended in an acetonitrile: water (7:3) solution buffered to pH = 2.5 using O.1M potassium hydogenpthalate and HCl acid, at a concentration of 1mg/ml, and sonicated for 10 minutes.20 The requisite This journal is © The Royal Society of Chemistry [year]
Fig 2. Powder XRD (a) and FT-IR (b) analysis of NBD-AP-MCM.
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concentration of aqueous fluoride ion or other ions were then added to this suspension, and stirred for 2 hours (except for the time dependent study) at room temperature. Finally, this dispersion was directly introduced to a cuvette and used for emission, absorption or lifetime measurements. e) Sample preparation for cellular uptake of NBD-AP-MCM: Tetrahymena thermophila cells were grown in SPP media (2% proteose peptone, 0.2% dextrose, 0.1% yeast extract,0.003% ferric EDTA) and when the cell number reached log phase , cells were starved in DMC media (0.17 mM sodium citrate, 0.1 mM NaH2PO4, 0.1 mM Na2HPO4, 0.65 mM CaCl2, 0.1 mM MgCl2) for 16 hours. Starved cells were incubated for 4 hours with 100 μg/ml NBD-AP-MCM and other with 100 μg/ml NBD-AP-MCM with 10-4M tetrabutylammonium fluoride concentration. After 4 hours of incubation cells were fixed in 4% paraformaldehyde, washed twice with 10 mM HEPES (pH 7.5) and visualized under fluorescent microscope. f) Characterization: FE-SEM images (Figure 1) of NBD-APMCM show the aggregation of silica particle, giving rise to bigger aggregates with irregular shape and size. Surface of these particles display a porous morphology. Energy-dispersive X-Ray (EDX) spectroscopy was employed for elemental analysis of the sample (Figure 1), which revealed the presence of 5% nitrogen and 20% carbon, by weight, which is attributed to the presence of surface immobilized propylamine groups and NBD groups, connected by the propylamine spacer. 28 Powder XRD pattern shows a signal around 2θ = 2.21°, Journal Name, [year], [vol], 00–00 | 3
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Fig 3. Thermogravimetric analysis of AP-MCM (Red) and NBD-AP-MCM (Blue) particles, to measure decomposition of surface functionalization. 5
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characteristic of the mesoporous structure (Figure 2) ,but, a reduction in peak intensity and considerable broadening of the peak, compared to unmodified MCM-41, was observed, denoting the distortion of mesoporous structure, due to the organic modification of the inner walls of mesoporous structure, by the probe moieties.29 FTIR is used to examine the linkage of NBD to MCM-41 surface and general MCM-41 bonding patterns (Figure 2). The pronounced signals in the range 3690-3220 cm-1 corresponding to the surface silanol groups and 1350-840 cm-1 corresponding to the lattice Si-O-Si vibration, confirms the integrity of the basic silica framework.30 Bands at about 2850-2950 arise due to the
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alkyl group (-CH2-) of the aminopropylsilyl linker between the fluorophore and the mesoporous silica surface. Band at 1600 cm1 can be attributed to the presence of NO2 of the NBD moiety. Most of the bands corresponding to NBD moiety have been masked by the more intense bands of MCM-41. Thermogravimetric analysis of both NBD-AP-MCM and APMCM were carried out to obtain the amount of postgrafted NBD on MCM-41 surface (Figure 3). A loss in weight of both the samples can be observed at around 70°C, which indicates the loss of physically absorbed water. The loss is less prominent for NBD-AP-MCM, since the additional presence of the fluorophore reduces the volume available for water to be absorbed. Subsequently, a decrease of 10.72% weight of the sample was observed in AP-MCM sample from 350°-600°C, which is attributed to the surface coverage 0.8 milimoles 3-aminopropyl group per gram AP-MCM , and 20.66% in NBD-AP-MCM, from 150°-700°C, and is attributed to all the organic functionalities. Since we know that 10.72% weight loss is due to the 3aminopropyl group, 9.348% of the sample weight can be attributed to NBD probe, which equals 0.56 milimoles of NBD molecules, per gram postgrafted mesoporous material.
4. Result and Discussions
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NBD is known to exhibit a broad absorption and fluorescence band typical of the intramolecular charge transition from amino nitrogen to nitro-group of NBD fluorophore moiety. 25 NBD is widely used in cell biology for cellular imaging applications. 31,32 Most of these applications stem on its remarkable environment sensitivity, due to strong quenching of fluorescence and
Fig 4. NBD-AP-MCM and NBDH in acetonitrile: water (7:3) solvent (pH=2.5) ; a) Absorption Spectra; b) Emission Spectra (λ exc . = 460 nm) ; c) Fluorescence Decay Profile (λexc. = 405 nm and λ emi. = 538 nm), where τav (NBD-AP-MCM) = 0.80 ns and τav (NBDH) = 2.75 ns.
considerable decrease in lifetime of the probe in aqueous environment ( less than 1 ns compared to 7-10 ns in low polar solvent)33. This discrimination in its behaviour towards polar solvents derives from its hydrogen bonding interactions with polar protic solvents, leading to increase in the rate of nonradiative decay, which in turn quenches fluorescence and decreases lifetime.33 NBD probes have been tagged to various biomolecules, in order to study transition between hydrophobic and hydrophilic environment of the biomolecules. 31,32 In the present work, we have postgrafted the NBD probe to the surface 4 | Journal Name, [year], [vol], 00–00
of MCM-41, via an APTES linkage, and the photophysics of the resulting system NBD-AP-MCM was investigated to analyse its anion sensing ability. 60
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(a) Photophysical properties of NBD-AP-MCM in absence and presence of Anions: The absorption behaviour of NBD-AP-MCM, suspended in an acetonitrile/water solvent system (7:3), is observed to be considerably different from that an unattached NBD probe. Even though there is very small shift in the position of absorption This journal is © The Royal Society of Chemistry [year]
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Fig 5. Absorption spectra of NBD-AP-MCM particles (1mg/ml) upon addition of increasing concentration of tetrabutylammonium fluoride, in acetonitrile: water (7:3) solvent (pH=2.5). 5
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maxima, but a noticeable broadening of the absorption band was observed (Figure 4). The full width at half maxima (FWHM) corresponding to unbound NBD, NBDH (7nitrobenzo[c][1,2,5]oxadiazol-4-amine) and bound NBD are estimated to be 67 nm and 108 nm. The observation indicates that that the probe postgrafted at the surface of mesoporous silica, undergoes aggregation on the mesoporous silica surface.34 The effects of aggregation of fluorophores on their photophysical properties has been extensively studied, in the solution phase. 35-37 Here, aggregation may be a result of π-π stacking interactions among several NBD moieties that are bound to the silica surface.34 Nevertheless, the overall absorption feature of both bound and unbound NBD indicates that the basic chromophoric properties of NBD are not significantly altered upon covalent attachment to mesoporous silica surface. During steady state fluorescence measurements the spectral profile remains very similar (Figure 4) for both bound and unbound NBD. However, significant decrease in the fluorescence quantum yield of the fluorophore in NBD-AP-MCM has been observed as compared to the unbound NBD molecule (Figure 4). The observation clearly points towards the presence of Aggregation Caused Quenching (ACQ), a phenomenon widely observed in case of aggregates of fluorescent molecules. 30,34,38 We have also carried out time-resolved studies on bound and unbound NBD systems. The decay profiles are shown in Figure 4. Initially, the average lifetime of the sensor (NBD-AP-MCM ) in acetonitrile/water (7:3) is estimated to be 0.80 ns, the value is significantly lower than the lifetime of NBD in the same solvent system (2.75 ns). This provides an evidence for the fact that the fluorescence quenching is not merely the result of interaction of NBD with the aqueous environment, but mainly due to aggregation caused quenching of NBD fluoroprobes MCM-41 surface. It may be mentioned here that in this (ACQ) process, excited state of a molecule is relaxed to the ground state via vibrational relaxation processes, as a result of hydrogen bonding or π-π interactions between the aromatic molecules that are in close proximity to each other. 34,39-41 Moreover, the electron
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Fig 6. Photoluminescence spectra of NBD-AP-MCM particles (1mg/ml) upon addition of tetrabutylammonium fluoride, in acetonitrile: water (7:3) solvent (pH=2.5). Inset shows a plot of emission intensity as a function of fluoride ion concentration (λ exc. = 460 nm).
transfer or the energy transfer between the molecules also causes the reduction in fluorescence intensity and lifetimes of the fluorophores that are attached to silica surface. 39-41 Further, to investigate the interaction of different commonly encountered anions with the present system NBD-AP-MCM, the steady state absorption and fluorescence measurements have been carried out. As can be seen from figure 5 upon gradual addition of fluoride ions the peak position (460 nm) of the absorption band remains more or less same. Interestingly, with gradual addition of F¯, the fluorescence intensity of the system is considerably enhanced, thereby signalling the presence of fluoride ions. In fact, more than 6 fold fluorescence enhancement upon addition 10 -3 M fluoride ion (Figure 6) was observed. The sample is excited at wavelength where change in absorbance due to F¯ is minimal. Further, the error in fluorescence intensity measurement, that may be caused due to change in optical density, is also taken care of, by carrying out a blank measurement. At this juncture, it is also interesting to note that fluoride, which is known in literature to behave as a quencher of NBD fluorescence primarily due to hydrogen bonding interaction between NBD and F¯42,43 is facilitating the fluorescence enhancement of the same. This apparent inconsistency in the behaviour of fluoride with the present silica-based material, in fact, indicates that some different kind of intermolecular interaction is taking place between the system and fluoride. As can be seen from Figure 6, that as the concentration of fluoride increases, fluorescence enhancement is observed till a fluoride concentration of 10-3 M, at which, the fluorescence signal gets saturated. It can be noted, that the peak position shows no alteration, illustrating that no change in molecular composition or complexation with fluoride ion, is responsible for enhancement of fluorescence. Additionally, the absorption band gradually decreases in width with increasing concentration of fluoride, progressively resembling that of unbound NBD species, indicating an increase in concentration of unbound NBD in the solution (Figure 6). All these spectroscopic observation clearly indicates that NBD moiety is becoming free in solution after F¯ addition. A supporting evidence for the presence of unbound NBD moiety has been obtained from mass spectrometry also, Journal Name, [year], [vol], 00–00 | 5
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Fig 7. Fluorescence Decay profile of NBD-AP-MCM particles (1mg/ml) upon addition of tetrabutylammonium fluoride in acetonitrile: water (7:3) solvent (pH=2.5). λ exc. = 405 nm and λ emi. = 538 nm.
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Table 1 Average fluorescence lifetime at different fluoride concentration at 1mg/ml probe in Acetonitrile/water (7:3) at pH 2.5
Scheme 2. Fluoride attack on NBD postgrafted MCM-41(NBD-AP-MCM) and subsequent liberation of NBD to the aqueous/organic solvent.
Fluoride
a1
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a2
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where the dispersion of NBD-AP-MCM and fluoride, stirred for two hours, was centrifuged to precipitate the mesoporous silica particles, and the supernatant was analysed by mass spectrometry. The evidence of a mass fragment corresponds to the NBD-NH(CH2)2CH2Na moiety, i.e. unbound NBD attached to 3aminopropyl group, (since C-Si bond is susceptible to cleavage in mass spectrometer44) is observed. Also, it is evident from the fluorescence spectra (Figure 6), that, even a concentration of 10-5 M is sufficient to evoke a response of nearly threefold fluorescence enhancement. Many reports in the literature have concluded that fluoride has a very high affinity to attack Si-O bonds, compared to other anions, and can effectively induce silyl cleavage2,4-7. Correlating the observation in the present case and the literature evidence, the enhancement of fluorescence can be reasonably attributed to the cleavage of Si-O bonds, which binds the NBD and MCM-41 together, upon the nucleophilic attack of fluoride anion, leading to the escape of the fluorophore from the surface of MCM-41, to the solvent system, liberating the fluorophore (NBD), the process which in turn helps in facilitating the recovery of NBD fluorescence in the solvent system (Scheme 2). To investigate the system-analyte interaction further, the time resolved fluorescence decay studies have been carried out. As stated earlier, the lifetime of the free sensor in acetonitrile/water (7:3) has been estimated to be 0.80 ns, which is significantly lower than the lifetime of NBD in the same solvent system (2.75 ns). The lifetime of the system has been monitored as a function of concentration of fluoride ion added to the system. The representative decay profiles of the system at various stages of F¯ concentration are shown in figure 7. The decay parameters are collected in Table 1. As can be seen from figure 7, a continuous increase in lifetime of the system from 0.80 ns to 2.46 ns, has been observed upon gradual addition of F¯ to the present system. Interestingly, as can be seen with the increase in concentration of the fluoride, the lifetime increases and final value of lifetime of the system at saturation level becomes very close to the lifetime
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τav, represent the average lifetime. τ1, τ2, τ3 and a1, a2, a3 represent the exponential fitting life time values and their contribution to average lifetime respectively.
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value of the unbound probe in the same solvent system (2.75 ns) illustrating the fact that NBD recovers its original fluorescence that was quenched due to aggregation on silica surface. To evaluate the response time required for fluoride ion detection in a sample, we have performed a time-dependent steady state study of the variation in fluorescence intensity of the system, at 5x10-5 M fluoride ion concentrations in 1mg/ml solution of NBDAP-MCM (Figure 8). The fluorescence of the reaction mixture has been measured at various intervals of time, ranging from 0 to 380 minutes. During the investigation, a continuous enhancement in the sensor fluorescence has been observed (Figure 8). As can be seen from the curve that nearly 3 fold fluorescence increases is observed in the first 40 minutes of reaction, which is sufficient to signal the presence of fluoride in a solution, hence, the response time of the system can be assigned to be as low as 40 minutes. It is evident from the constant increment of fluorescence, that fluoride is gradually able to diffuse through the mesoporous material, resulting in more NBD moieties being liberated from the solid surface, and add to system's fluorescence. The fluorescence enhancement saturates at about 240 minutes after fluoride addition, at which, the fluorescence has enhanced by nearly 6-7 fold of the initial value.
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Fig 8. Time dependent photoluminescence spectra of NBD-AP-MCM -5 particles (1mg/ml) upon attack by 5x10 M tetrabutylammonium fluoride, in acetonitrile: water (7:3) solvent (pH=2.5), from 0 minutes (black) to 380 minutes. Inset shows a plot of emission intensity as a function of time (λexc. = 460 nm).
Fig 10. Photoluminescence intensity of NBD-AP-MCM particles (1mg/ml), -4 upon attack by 1x10 M tetrabutylammonium fluoride, in acetonitrile: water (7:3) solvent, as a function of pH of the solution (λ exc. = 460 nm, λemi. = 541 nm). 30
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Fig 9. Change in fluorescence enhancement of NBD-AP-MCM (1mg/ml) -5 system upon addition of 5x10 M of tetrabutylammonium salt of various anions in acetonitrile: water (7:3) solvent (pH=2.5). Inset shows the emission plots corresponding to the bar graph (λexc. = 460 nm).
It may be noted here that the system is able to exhibit high fluorescence intensities even for low concentration of fluoride if sufficient time is given. As a result, the observation also gives us a clue to how to fine tune the sensitivity of the sensor system in terms of response time. Now, it can be easily inferred from the above study on the mechanism of fluoride signalling by NBD-AP-MCM, that the event of fluoride detection is a result of a specific reaction, particular to fluoride ion only, and hence the probe is expected to show high selectivity towards fluoride ion. Fluoride is known to have very high affinity to attack Si-O bonds, compared to other anions2,4-7. Fluoride is the most electronegative of all elements, and it has the least size among all comparable anions. Hence the
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effect produced by a chemical moiety with such a unique charge density, cannot be replicated by other anions. This is the key factor which enables the sensor to distinctively identify the fluoride anion. Selectivity of NBD-AP-MCM towards fluoride has been assayed against chloride, bromide, iodide, acetate and bisulphate anions using 5x10-5 M of each ion, in 1mg/ml suspension of NBD-AP-MCM and stirring for two hours, to determine the effect of these competing anions on the sensor fluorescence (Figure 9). As expected, the present sensor system have exhibited a remarkable discrimination between fluoride and other ions, displaying a considerable fluorescence enhancement upon fluoride addition, whereas an insubstantial change in fluorescence has been observed in the cases of other anions (Figure 9). The fluoride sensing experiments reported in this paper, are conducted at pH = 2.5, to maximize the sensitivity of fluoride detection. We have also investigated the fluorescence response of the system in absence and presence of fluoride at various other pH. In absence of fluoride fluorescence response of the present systems is observed to be not affected significantly (see supplementary material). However, a significant change has been observed while monitoring the signalling event of the system in presence of fluoride (Figure 10). From the figure, it is clear that 2.5 to 3.5 is the range of pH, where fluoride signalling event takes place with maximum efficiency. The observation is consistent with literature report where Si-O bond cleavage has been observed at low pH (~2.5). 20We would like also like mention that in pure water the change (fold) in fluorescence enhancement has been measured to very low (1.2-1.5), with no noticeable change in lifetime. This observation is not surprising when we take into account the fact that, both fluorescence quantum yield and lifetime of NBD systems are observed to be very low in pure water.25,33 The increase in non-radiative rate constants due to hydrogen bonding interaction between the NBDbased system and water is primarily responsible for low fluorescence yield and excited state lifetime of the system. 25,33 However, upon increasing the concentration of organic solvent (acetonitrile), in the medium to 7:3 (acetonitrile : water, v/v), the lifetime significantly increased to 2.7 ns. Cellular uptake studies reveal that the system is not toxic to cells and can be used in cellular imaging purposes (see supplementary material).
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A 7-nitro-2,1,3-benzoxadiazole (NBD) anchored mesoporous silica system, NBD-AP-MCM, was synthesized, and characterized using powder-XRD, FE-SEM, EDX, FTIR and Thermogravimetric analysis. The system was shown to sensitively and selectively discriminate fluoride in an aqueous/organic (3:7, pH = 2.5) solvent system. Initially present in completely quenched state, due to aggregation caused quenching (ACQ) of fluorophore, the system displayed up to six fold fluorescence enhancement upon fluoride attack, which is attributed to a specific Si-O bond cleavage reaction, leading to transfer of fluorophore to the organic/aqueous solvent system and subsequent regaining of fluorescence. The working range of concentration for fluoride sensing was estimated to be 10 -5 M to 10-3 M, and the response time can be as low as 40 minutes. The workable pH conditions are pH = 2.3 to 3.5 for sensitive measurement. Further steady state and time dependent studies have been carried out to confirm and explore the mechanistic details of transition of fluorophore from solid to solution phase. Additionally, cellular uptake studies are conducted (see supplementary material) to evaluate the system's biocompatibility, and its application in cellular imaging studies.
Acknowledgement 25
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M.S. thanks the National Institute of Science Education and Research (NISER), Bhubaneswar for funding. G.J. is thankful to the Department of Science and Technology (DST), New Delhi for providing the Kishore Vaigyanik Protsahan Yojana (KVPY) fellowship. A.N. is thankful to Council of Scientific and Industrial Research (CSIR), New Delhi, for the fellowship awarded to him.
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Notes and references a
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School of Chemical Sciences, National Institute of Science Education and Research, Bhubaneswar 751005, India. Fax: +91-674-2302436 Tel: +91-674-2304037 Email: [email protected] b School of biological Sciences, National Institute of Science Education and Research, Bhubaneswar 751005, India 1. L. H. Weinstein, A. Davison, Fluorides in the environment: effects on plants and animals, CABI, 2003. 2. Y. Zhou, J.F. Zhang, J. Yoon, Chem. Rev. 2014, 114, 5511. 3. C. R. Wade, A. E. J. Broomsgrove, S. Aldridge, F. P. Gabbaï, Chem. Rev. 2010, 110, 3958. 4. X. Li, X. Gao, W. Shi, H. Ma, Chem. Rev. 2014, 114, 590. 5. T. H. Kim, T. M. Swager, Angew. Chem. Int. Ed. 2003, 42. 4803. 6. P. Sokkalingam, C. H. Lee, J. Org. Chem. 2011, 76, 3820. 7. R. Hu, J. Feng, D. Hu, S. Wang, S. Li, Y. Li, G. A. Yang, Angew. Chem. Int. Ed. 2010, 49, 4915. 8. D. E. Gómez, L. Fabbrizzi, M. Licchelli, J. Org. Chem. 2005, 70, 5717. 9. J. Shao, H. Lin, H. K. Lin, Talanta 2008, 75, 1015. 10. S. Rochat, K. Severin, Chem. Commun. 2011, 47, 4391. 11. P. Thiampanya, N. Muangsin, B. Pulpoka, Org. Lett. 2012, 14, 4050. 12. T. Agou, M. Sekine, J. Kobayashi, T. Kawashima, Chem. Commun. 2009, 1894. 13. S. V. Bhosale, M. B. Kalyankar, S. J. Langford, Org. Lett. 2009, 11, 5418. 14. O. M. Cabarcos, C. J. Weinheimer, J. M. Lisy, S. S. Xantheas, J. Chem. Phys. 1999, 110, 5 15. S. Y. Kim, J. I. Hong, Org. Lett. 2007, 9, 3109-3112. 16. S. Y. Kim, J. Park, M. Koh, S. B. Park, J. I. Hong, Chem. Commun. 2009, 4735.
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17. L. B. Desmonts, D. N. Reinhoudt, M. C. Calama, Chem. Soc. Rev. 2007, 36, 993. 18. B. J. Scott, G. Wirnsberger, G. D. Stucky, Chem. Mater. 2001, 13, 3140. 19. J. L. Shi, Z. L. Hua, L. X. Zhang, J. Mater. Chem. 2004, 14, 795. 20. A. B. Descalzo, D. Jiménez, J. E. Haskouri, D. Beltrán, P. Amorós, M. D. Marcos, R. Martínez-Máñez, J. Soto, Chem. Commun. 2002, 562. 21. H. Meng, M. Xue, T. Xia, Y. L. Zhao, F. Tamanoi, J. F. Stoddart, J. I. Zink, A. E. Nel, J. Am. Chem. Soc. 2010, 132, 12690. 22. M. Vallet-Regí, F. Balas, D. Arcos, Angew. Chem. Int. Ed. 2007, 46, 7548. 23. I. I. Slowing, J. L. Vivero-Escoto, C. W. Wu, V. S. Y. Lin, Adv. Drug Delivery Rev. 2008, 60, 1278. 24. X. Xinjian Yang, Z. Li, M. Li, J. Ren, X. Qu, Chem. Eur. J. 2013, 19, 15378. 25. S. Saha, A. Samanta, J. Phys. Chem. A. 1998, 102, 7903. 26. A. J. Boulton, P. B. Ghosh, A. R. Katritzky, J. Chem. Soc. B, 1966, 1004. 27. H. Ritter, M. Nieminen, M. Karppinen, D. Brühwiler, D. Microporous and Mesoporous Mater. 2009, 121, 79. 28. S. M. Alahmadi, S. Mohamad, M. J. Maah, Adv. Mat. Sci. Eng. 2013, 8. 29. B. Marler, U. Oberhagemann, S. Vortmann, H. Gies, Microporous Mater. 1996, 6, 375. 30. F. J. Trindade, J. F. Q. Rey, S. Brochsztain, Dyes and Pigments. 2011, 89, 97. 31. A. Chattopadhyay, Chem. Phys. Lipids 1990, 53, 1. 32. S. Mazeres, V. Schram, J. F. Tocanne, A. Lopez, Biophys. J. 1996, 71, 327. 33. S. Lin, W. Struve, Photochem. Photobio. 1991, 54, 361. 34. I. Candel, P. Calero, R. Martínez-Máñez, F. Sancenón, M. D. Marcos, T. Pardo, J. Soto, Inorg. Chim. Acta 2012, 381, 184-194. 35. M. Soni, S. K. Das, P. K. Sahu, U. P. Kar, A. Rahaman, M. Sarkar, J. Phys. Chem. C, 2013, 117 (27),14338. 36. D. Majhi, S. K. Das, P. K. Sahu, S. M. Pratik, A. Kumar, M. Sarkar, Phys. Chem .Chem. Phys. 2014, 16, 18349. 37. C. T. Chen, Chem. Mater. 2004, 16, 4389. 38. F. J. Trindade, J. F. Q. Rey, S. Brochsztain, J. Colloid Interface Sci. 2012, 368, 34. 39. A. Dreuw, J. Plötner, L. Lorenz, J. Wachtveit, J. E. Djanhan, J. Brning, T. Metz, M. Bolte, M. Schmidt, Angew. Chem. Int. Ed. 2005, 44, 7783. 40. B. M. Aveline, T. Hasan, R. W. Redmond, J. Photochem. Photobiol. B 1995, 30, 161. 41. W. Z. Yuan, P. Lu, S. Chen, J. W. Y. Lam, Z. Wang, Y. Liu, H. S. Kwok, Y. Ma, B. Z. Tang, Adv. Mater. 2010, 22, 2159. 42. A. K. Bhoi, S. K. Das, D. Majhi, P. K. Sahu, A. Nijamudheen, N. Anoop, A. Rahaman, M. Sarkar, J. Phys. Chem. B 2014, 118, 9926. 43. S. K. Das, S. S. Misra, P. K. Sahu, A. Nijamudheen, V. Mohan, M. Sarkar, Chem. Phys. Lett. 2012, 546, 90. 44. V. Zaikin, J. M. Halket, A Handbook of Derivatives for Mass Spectrometry, IM Publications, 2009.
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Fluoride Ion Sensing in Aqueous Medium by Employing Nitrobenzoxadiazole-Postgrafted Mesoporous Silica Nano Particles (MCM-41) Gaurav Jhaa, Anoop N. b, Abdur Rahamanb and Moloy Sarkar*a 5
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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x Mesoporous Silica-based inorganic-organic hybrid material (NBD-AP-MCM) as fluorescent chemosensor for detection of fluoride in aqueous medium has been designed and developed. The system has been developed by covalently anchoring 7-nitro-2, 1, 3-benzoxadiazole (NBD) dye to the surface of mesoporous silica nano particles, MCM-41. The system has been characterized by several conventional analytical methods comprising spectroscopic, microscopic and thermo-gravimetric techniques. Sensory action of the material has been investigated by carrying out steady state absorption and fluorescence and time resolved fluorescence studies on the system in absence and presence of several biologically and environmentally important anions in aqueous solution. Photophysical data of the present system (NBDAP-MCM) are also compared with the free dye (NBD) molecule. Significant decrease in the fluorescence quantum yield of the fluorophore in the hybrid material NBD-AP-MCM has been observed as compared to the unbound NBD. The decrease in fluorescence efficiency in hybrid material is attributed to the aggregation caused quenching (ACQ) phenomenon. Interestingly, the system displays more than six fold fluorescence enhancement in presence of fluoride ion in aqueous solution. Enhancement of fluorescence lifetime of the fluorescing moiety (NBD) has also been observed during fluorescence time-resolved studies. No significant optical changes have been observed with other commonly encountered anions rendering the present system highly selective towards fluoride detection. The fluorescence enhancement has been attributed to the cleavage of Si-O bonds due to the addition of fluoride. The silyl cleavage detaches the fluorophore from the solid support and thereby making the fluorophore “free” in solution which in turn recovers its original fluorescence which was decreased because of the aggregation on solid silica support. Furthermore, the suitability of the present system in cellular imaging has also been demonstrated.
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Fluoride is an essential anion, abundantly present in soil, ocean and ground water1. Its utility in prevention of dental caries, and treatment of osteoporosis, has been explored widely2. Fluoride ions are also detected as an indicator of uranium refinement (via hydrolysis of UF 6) or of the chemical warfare agent sarin, which releases fluoride ion upon hydrolysis. 2 However, overexposure to fluoride is harmful for biological systems and in human beings, it causes nausea, fluorosis, abdominal pain, coma, cardiac arrest etc.2,3 In the light of these problems, development of fluoride detection techniques have gained widespread attention in recent years, especially using fluorescence technique, which allows high sensitivity, low detection limit and is capable of intracellular fluoride detection.2-4 The chemosensors reported in literature, often utilize fluoride promoted bond cleavage,4-7 boron−fluoride interaction3, This journal is © The Royal Society of Chemistry [year]
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hydrogen-bond and π−π interaction8,9, and metal binding10,11. But majority of the chemosensors reported, require organic medium, since aqueous medium poses problems regarding solubility, polarity and hydrogen bonding. 12,13 Moreover, one inherent problem for detection of fluoride ion in aqueous medium is due its high hydration energy. 14 It may be mentioned that sensors discriminating through fluorescence quenching are prone to lower selectivity of sensing as compared to those based on fluorescence enhancement. Very few probes have been reported in literature, which actively sense fluoride without these restrictions, 6,15,16 for practical application of fluoride detection in aqueous solution. Moreover, most sensors are still too large for a number of conceivable applications and miniaturization to the nanoscale is essential for widespread applicability. Hence, development of adequate nanomaterials, is an important direction for sensor technologies. One of the interesting physical attributes of nano
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Scheme 1. Schematic representation of postgrafting of 7-nitrobenz-2oxa-1,3-diazol-4-yl (NBD) probe on MCM-41 surface. 5
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particles is their ability to provide a much larger surface to volume ratio compared to that of bulk material. Decreasing the size of the sensor has several advantages, which include the need for assaying smaller sample volumes, lowering available detection limits and reducing the amount of the involved sensing chemicals. In view of this, it is highly important to develop new sensing methodologies by exploiting nanoparticle –based sensor systems towards a simple, inexpensive, sensitive and selective detection of fluoride ion in aqueous medium. Mesoporous silica materials (e.g. MCM-41) are emerging as a new versatile, non-toxic and water dispersible solid support for immobilization of fluorogenic probes on their surface for optical sensing device fabrication17,18. The characteristic properties of mesoporous silica materials, such as high surface area which allows large probe doping concentrations, and highly uniform porosity enabling facile diffusion of analyte molecules, renders these materials to be an excellent support for photoluminescent sensing probes19,20. Such mesoporous material-fluorophore conjugates are widely used for small molecule and ion sensing, drug-delivery and in-vivo cellular imaging purposes. 21 Compared to free dye molecules, which are prone to photobleaching, solvatochromic effects and often display cellular toxicity, mesoporous silica based fluorescent probes are highly biocompatible, demonstrate enhanced per particle brightness and there surface can be easily functionalized for biological targeting by anchoring proteins, antibodies or cell penetrating peptides. 22-24 The optical transparency in UV and visible range, 18,20 of these materials, enables the exploration of probe behaviour on solid support as well as their transition from solid to solution phase. 2 | Journal Name, [year], [vol], 00–00
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There is an additional requirement of an environment sensitive probe, to signal the event of this phase transition from solid to solution, upon the attack of an analyte moiety detaching the probe from the solid support. Such a system can selectively discriminate between analytes, based on specific reactions, and give rise to a highly selective optical sensor that is capable of detecting analytes in aqueous medium at low detection limit. In the present work, we have devised a new fluoride anion sensor, consisting of a 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) probe, covalently anchored to MCM-41 surface. NBD probe has been covalently grafted on MCM-41 surface by means of 3(aminopropyl)triethoxysilane, in order to prevent the leaching of dye molecules, in solution phase17. NBD is chosen as the signalling fluorophore for this system, due to its charge transfer based highly robust fluorescence, environment sensitivity and the ease of reaction pertaining to probe labelling, which has been extensively exploited, both in vitro and in vivo applications. 25 The NBD moiety is in quenched state when attached to the MCM-41 surface in acetonitrile/water (7:3) mixture. Fluoride ion exhibits a nucleophilic attack on the Si-O bond, which is responsible for holding together the silica surface and the fluorophore, thereby releasing the NBD moiety into the aqueous/organic solvent system, where the probe, regains its fluorescence and signals the presence of fluoride through an enhancement in fluorescence. This transition of NBD from solid support to solution has been monitored via steady state and time dependent fluorescence studies. The affinity of fluoride ion for Si-O and Si-C bonds has been extensively studied and utilized for design of fluoride ion sensing probes where fluoride attack leads to generation of more fluorescent chemical species, 2,4-7 mostly anions, but these probes require the resulting anions to be in conjugation with the fluorogenic system. Designing such conjugated systems require extensive organic synthesis and tricky purification steps. However, the sensor design using postgrafting of mesoporous silica requires nominal synthesis and easy purification, with considerably higher yields. Furthermore, this work has been inspired form the considerable deficit of studies in the literature, focusing on the detailed photophysical analysis of the behaviour of ion sensing probes, postgrafted on the solid surface of mesoporous silica particles. These studies are important to provide better understanding of the mechanisms of ion sensing and dynamics of the probe at the silica surface, and to further devise probes having better sensitivity, selectivity and simpler construction 20.
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The absorption and emission spectra are recorded on a UVvisible spectrometer (Cary100, Varian) and a spectrofluorimeter (Perkin Elmer, LS 55), respectively. The fluorescence spectra are corrected for instrumental response. Time resolved fluorescence measurements are carried out by a time-correlated single photon counting (TCSPC) spectrometer (Edinburg, OB920). Picosecond pulse diode laser (EPL-405) is used as excitation source, and an MCP photomultiplier (Hamamatsu R3809U-50) is used as the detector (response time 40ps). The instrumental response time of our instrument is about 98 ps (FWHM). The lamp profile is recorded by scaterer (dilute ludox solution in water) in place of This journal is © The Royal Society of Chemistry [year]
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the sample. Elemental analysis through EDX (energy-dispersive X-ray spectroscopy) and scanning electron microscopy (SEM) images of postgrafted mesoporous silica were obtained using field-emission scanning electron microscope (FESEM) system (Carl Zeiss, Germany make, model: Pigma). The SEM samples were prepared by drop casting dispersion of postgrafted mesoporous silica in acetonitrile: water (7:3), and evaporating in air at room temperature. Powder X-ray diffraction (p-XRD) patterns were performed on a Bruker DAVINCI D8 ADVANCE diffractometer equipped with Cu Ka radiation (l ¼ 0.15406 nm). FT-IR measurements of the system were performed using a Perkin-Elmer Spectrum RXI FT-IR spectrophotometer, and thermogravimetric analysis (TGA) was carried out using PerkinElmer Simultaneous Thermal Analyzer STA-6000. Fluorescence microscopy images were taken in Axio Imager.M2 at 40X magnification, detected with filter AF 488. Mesostructured silica, MCM-41 type, 4-chloro-7nitrobenzofurazan (98%), sodium azide (>99.5%), tetrabutylammonium fluoride hydrate (98%), tetrabutylammonium chloride (>97.0%), tetrabutylammonium bromide (>98%), tetrabutylammonium iodide (purum >98%), tetrabutylammonium acetate (>97%) and tetrabutylammonium bisulphate(purum, >97%) were obtained from Sigma-Aldrich and were used without any further purification. 3-aminopropyl triethoxysilane(APTES) was obtained from Himedia and was used without any further purification. Acetonitrile used in photophysical studies was anhydrous and was purchased from Sigma-Aldrich.
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Fig 1. FE-SEM images of NBD-AP-MCM (upper panel) scale bar = 200 nm, EDX micrograph (lower panel, left side) and elemental composition of NBD-AP-MCM (lower panel, right side). ). Elemental Composition : Carbon (C)=19.9%, Nitrogen (N)=5.0%, Oxygen(O)= 37.7%, Silicon(Si) = 37.7%.
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a) Synthesis of NBDH: NBDH (7-nitrobenzo[c][1,2,5]oxadiazol4-amine) was prepared from 4-chloro-7-nitrobenz-2-oxa-1,3diazole (NBD-Cl) by following a standard procedure. 26 The compound was characterized by 1H NMR. Yield of NBDH ~ 50%. 1H NMR of NBDH (DMSO-d6, ppm): δ 8.89 (s, 2H), 8.52 (d, 1H), 6.42 (d, 1H). b) Synthesis of AP-MCM: APTES (3-aminopropyl triethoxysilane) was anchored to MCM-41, using a reported procedure 27. 500 mg of MCM-41 was pre-treated at 180 °C for 2 h and dispersed in 30 mL of dry toluene. After the addition of APTES (160 μL for the targeted functionalization degree of 1.2 mmol per gram of parent MCM-41), the suspension was refluxed for 3 h. The functionalized product was recovered by filtration, washed with 100 mL of ethanol, and oven-dried at 80 °C for 1 h. c) Synthesis of NBD-AP-MCM: 100 mg of dried AP-MCM was dispersed in 10ml ethanol. 100 mg (0.5 mmol) of 4-chloro-7nitrobenzo[c][1,2,5]oxadiazole (NBD-chloride) was added to the solution and the resulting mixture was left stirring for 2-3 hours. The functionalized product was recovered by filtration, washed with ethanol, dichloromethane and water and oven-dried at 110 °C for 2 hours. d) Sample Preparation for Anion Sensing: A general procedure was followed to study the interaction of anions, with NBD-APMCM. The synthesized NBD-AP-MCM was suspended in an acetonitrile: water (7:3) solution buffered to pH = 2.5 using O.1M potassium hydogenpthalate and HCl acid, at a concentration of 1mg/ml, and sonicated for 10 minutes.20 The requisite This journal is © The Royal Society of Chemistry [year]
Fig 2. Powder XRD (a) and FT-IR (b) analysis of NBD-AP-MCM.
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concentration of aqueous fluoride ion or other ions were then added to this suspension, and stirred for 2 hours (except for the time dependent study) at room temperature. Finally, this dispersion was directly introduced to a cuvette and used for emission, absorption or lifetime measurements. e) Sample preparation for cellular uptake of NBD-AP-MCM: Tetrahymena thermophila cells were grown in SPP media (2% proteose peptone, 0.2% dextrose, 0.1% yeast extract,0.003% ferric EDTA) and when the cell number reached log phase , cells were starved in DMC media (0.17 mM sodium citrate, 0.1 mM NaH2PO4, 0.1 mM Na2HPO4, 0.65 mM CaCl2, 0.1 mM MgCl2) for 16 hours. Starved cells were incubated for 4 hours with 100 μg/ml NBD-AP-MCM and other with 100 μg/ml NBD-AP-MCM with 10-4M tetrabutylammonium fluoride concentration. After 4 hours of incubation cells were fixed in 4% paraformaldehyde, washed twice with 10 mM HEPES (pH 7.5) and visualized under fluorescent microscope. f) Characterization: FE-SEM images (Figure 1) of NBD-APMCM show the aggregation of silica particle, giving rise to bigger aggregates with irregular shape and size. Surface of these particles display a porous morphology. Energy-dispersive X-Ray (EDX) spectroscopy was employed for elemental analysis of the sample (Figure 1), which revealed the presence of 5% nitrogen and 20% carbon, by weight, which is attributed to the presence of surface immobilized propylamine groups and NBD groups, connected by the propylamine spacer. 28 Powder XRD pattern shows a signal around 2θ = 2.21°, Journal Name, [year], [vol], 00–00 | 3
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Fig 3. Thermogravimetric analysis of AP-MCM (Red) and NBD-AP-MCM (Blue) particles, to measure decomposition of surface functionalization. 5
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characteristic of the mesoporous structure (Figure 2) ,but, a reduction in peak intensity and considerable broadening of the peak, compared to unmodified MCM-41, was observed, denoting the distortion of mesoporous structure, due to the organic modification of the inner walls of mesoporous structure, by the probe moieties.29 FTIR is used to examine the linkage of NBD to MCM-41 surface and general MCM-41 bonding patterns (Figure 2). The pronounced signals in the range 3690-3220 cm-1 corresponding to the surface silanol groups and 1350-840 cm-1 corresponding to the lattice Si-O-Si vibration, confirms the integrity of the basic silica framework.30 Bands at about 2850-2950 arise due to the
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alkyl group (-CH2-) of the aminopropylsilyl linker between the fluorophore and the mesoporous silica surface. Band at 1600 cm1 can be attributed to the presence of NO2 of the NBD moiety. Most of the bands corresponding to NBD moiety have been masked by the more intense bands of MCM-41. Thermogravimetric analysis of both NBD-AP-MCM and APMCM were carried out to obtain the amount of postgrafted NBD on MCM-41 surface (Figure 3). A loss in weight of both the samples can be observed at around 70°C, which indicates the loss of physically absorbed water. The loss is less prominent for NBD-AP-MCM, since the additional presence of the fluorophore reduces the volume available for water to be absorbed. Subsequently, a decrease of 10.72% weight of the sample was observed in AP-MCM sample from 350°-600°C, which is attributed to the surface coverage 0.8 milimoles 3-aminopropyl group per gram AP-MCM , and 20.66% in NBD-AP-MCM, from 150°-700°C, and is attributed to all the organic functionalities. Since we know that 10.72% weight loss is due to the 3aminopropyl group, 9.348% of the sample weight can be attributed to NBD probe, which equals 0.56 milimoles of NBD molecules, per gram postgrafted mesoporous material.
4. Result and Discussions
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NBD is known to exhibit a broad absorption and fluorescence band typical of the intramolecular charge transition from amino nitrogen to nitro-group of NBD fluorophore moiety. 25 NBD is widely used in cell biology for cellular imaging applications. 31,32 Most of these applications stem on its remarkable environment sensitivity, due to strong quenching of fluorescence and
Fig 4. NBD-AP-MCM and NBDH in acetonitrile: water (7:3) solvent (pH=2.5) ; a) Absorption Spectra; b) Emission Spectra (λ exc . = 460 nm) ; c) Fluorescence Decay Profile (λexc. = 405 nm and λ emi. = 538 nm), where τav (NBD-AP-MCM) = 0.80 ns and τav (NBDH) = 2.75 ns.
considerable decrease in lifetime of the probe in aqueous environment ( less than 1 ns compared to 7-10 ns in low polar solvent)33. This discrimination in its behaviour towards polar solvents derives from its hydrogen bonding interactions with polar protic solvents, leading to increase in the rate of nonradiative decay, which in turn quenches fluorescence and decreases lifetime.33 NBD probes have been tagged to various biomolecules, in order to study transition between hydrophobic and hydrophilic environment of the biomolecules. 31,32 In the present work, we have postgrafted the NBD probe to the surface 4 | Journal Name, [year], [vol], 00–00
of MCM-41, via an APTES linkage, and the photophysics of the resulting system NBD-AP-MCM was investigated to analyse its anion sensing ability. 60
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(a) Photophysical properties of NBD-AP-MCM in absence and presence of Anions: The absorption behaviour of NBD-AP-MCM, suspended in an acetonitrile/water solvent system (7:3), is observed to be considerably different from that an unattached NBD probe. Even though there is very small shift in the position of absorption This journal is © The Royal Society of Chemistry [year]
Physical Chemistry Chemical Physics Accepted Manuscript
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Physical Chemistry Chemical Physics
Fig 5. Absorption spectra of NBD-AP-MCM particles (1mg/ml) upon addition of increasing concentration of tetrabutylammonium fluoride, in acetonitrile: water (7:3) solvent (pH=2.5). 5
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maxima, but a noticeable broadening of the absorption band was observed (Figure 4). The full width at half maxima (FWHM) corresponding to unbound NBD, NBDH (7nitrobenzo[c][1,2,5]oxadiazol-4-amine) and bound NBD are estimated to be 67 nm and 108 nm. The observation indicates that that the probe postgrafted at the surface of mesoporous silica, undergoes aggregation on the mesoporous silica surface.34 The effects of aggregation of fluorophores on their photophysical properties has been extensively studied, in the solution phase. 35-37 Here, aggregation may be a result of π-π stacking interactions among several NBD moieties that are bound to the silica surface.34 Nevertheless, the overall absorption feature of both bound and unbound NBD indicates that the basic chromophoric properties of NBD are not significantly altered upon covalent attachment to mesoporous silica surface. During steady state fluorescence measurements the spectral profile remains very similar (Figure 4) for both bound and unbound NBD. However, significant decrease in the fluorescence quantum yield of the fluorophore in NBD-AP-MCM has been observed as compared to the unbound NBD molecule (Figure 4). The observation clearly points towards the presence of Aggregation Caused Quenching (ACQ), a phenomenon widely observed in case of aggregates of fluorescent molecules. 30,34,38 We have also carried out time-resolved studies on bound and unbound NBD systems. The decay profiles are shown in Figure 4. Initially, the average lifetime of the sensor (NBD-AP-MCM ) in acetonitrile/water (7:3) is estimated to be 0.80 ns, the value is significantly lower than the lifetime of NBD in the same solvent system (2.75 ns). This provides an evidence for the fact that the fluorescence quenching is not merely the result of interaction of NBD with the aqueous environment, but mainly due to aggregation caused quenching of NBD fluoroprobes MCM-41 surface. It may be mentioned here that in this (ACQ) process, excited state of a molecule is relaxed to the ground state via vibrational relaxation processes, as a result of hydrogen bonding or π-π interactions between the aromatic molecules that are in close proximity to each other. 34,39-41 Moreover, the electron
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Fig 6. Photoluminescence spectra of NBD-AP-MCM particles (1mg/ml) upon addition of tetrabutylammonium fluoride, in acetonitrile: water (7:3) solvent (pH=2.5). Inset shows a plot of emission intensity as a function of fluoride ion concentration (λ exc. = 460 nm).
transfer or the energy transfer between the molecules also causes the reduction in fluorescence intensity and lifetimes of the fluorophores that are attached to silica surface. 39-41 Further, to investigate the interaction of different commonly encountered anions with the present system NBD-AP-MCM, the steady state absorption and fluorescence measurements have been carried out. As can be seen from figure 5 upon gradual addition of fluoride ions the peak position (460 nm) of the absorption band remains more or less same. Interestingly, with gradual addition of F¯, the fluorescence intensity of the system is considerably enhanced, thereby signalling the presence of fluoride ions. In fact, more than 6 fold fluorescence enhancement upon addition 10 -3 M fluoride ion (Figure 6) was observed. The sample is excited at wavelength where change in absorbance due to F¯ is minimal. Further, the error in fluorescence intensity measurement, that may be caused due to change in optical density, is also taken care of, by carrying out a blank measurement. At this juncture, it is also interesting to note that fluoride, which is known in literature to behave as a quencher of NBD fluorescence primarily due to hydrogen bonding interaction between NBD and F¯42,43 is facilitating the fluorescence enhancement of the same. This apparent inconsistency in the behaviour of fluoride with the present silica-based material, in fact, indicates that some different kind of intermolecular interaction is taking place between the system and fluoride. As can be seen from Figure 6, that as the concentration of fluoride increases, fluorescence enhancement is observed till a fluoride concentration of 10-3 M, at which, the fluorescence signal gets saturated. It can be noted, that the peak position shows no alteration, illustrating that no change in molecular composition or complexation with fluoride ion, is responsible for enhancement of fluorescence. Additionally, the absorption band gradually decreases in width with increasing concentration of fluoride, progressively resembling that of unbound NBD species, indicating an increase in concentration of unbound NBD in the solution (Figure 6). All these spectroscopic observation clearly indicates that NBD moiety is becoming free in solution after F¯ addition. A supporting evidence for the presence of unbound NBD moiety has been obtained from mass spectrometry also, Journal Name, [year], [vol], 00–00 | 5
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Fig 7. Fluorescence Decay profile of NBD-AP-MCM particles (1mg/ml) upon addition of tetrabutylammonium fluoride in acetonitrile: water (7:3) solvent (pH=2.5). λ exc. = 405 nm and λ emi. = 538 nm.
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Table 1 Average fluorescence lifetime at different fluoride concentration at 1mg/ml probe in Acetonitrile/water (7:3) at pH 2.5
Scheme 2. Fluoride attack on NBD postgrafted MCM-41(NBD-AP-MCM) and subsequent liberation of NBD to the aqueous/organic solvent.
Fluoride
a1
τ1(ns)
a2
τ2(ns)
a3
where the dispersion of NBD-AP-MCM and fluoride, stirred for two hours, was centrifuged to precipitate the mesoporous silica particles, and the supernatant was analysed by mass spectrometry. The evidence of a mass fragment corresponds to the NBD-NH(CH2)2CH2Na moiety, i.e. unbound NBD attached to 3aminopropyl group, (since C-Si bond is susceptible to cleavage in mass spectrometer44) is observed. Also, it is evident from the fluorescence spectra (Figure 6), that, even a concentration of 10-5 M is sufficient to evoke a response of nearly threefold fluorescence enhancement. Many reports in the literature have concluded that fluoride has a very high affinity to attack Si-O bonds, compared to other anions, and can effectively induce silyl cleavage2,4-7. Correlating the observation in the present case and the literature evidence, the enhancement of fluorescence can be reasonably attributed to the cleavage of Si-O bonds, which binds the NBD and MCM-41 together, upon the nucleophilic attack of fluoride anion, leading to the escape of the fluorophore from the surface of MCM-41, to the solvent system, liberating the fluorophore (NBD), the process which in turn helps in facilitating the recovery of NBD fluorescence in the solvent system (Scheme 2). To investigate the system-analyte interaction further, the time resolved fluorescence decay studies have been carried out. As stated earlier, the lifetime of the free sensor in acetonitrile/water (7:3) has been estimated to be 0.80 ns, which is significantly lower than the lifetime of NBD in the same solvent system (2.75 ns). The lifetime of the system has been monitored as a function of concentration of fluoride ion added to the system. The representative decay profiles of the system at various stages of F¯ concentration are shown in figure 7. The decay parameters are collected in Table 1. As can be seen from figure 7, a continuous increase in lifetime of the system from 0.80 ns to 2.46 ns, has been observed upon gradual addition of F¯ to the present system. Interestingly, as can be seen with the increase in concentration of the fluoride, the lifetime increases and final value of lifetime of the system at saturation level becomes very close to the lifetime
0M
0.078
0.01
0.041
1.23
0.011
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10-5 M
0.073
0.11
0.036
1.22
0.013
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0.85
10-4 M
0.014
0.30
0.010
1.47
0.015
3.76
1.95
10-3 M
0.025
0.37
0.037
2.44
0.016
5.48
2.40
10-2 M
0.042
1.71
0.029
3.53
0.000
7.82
2.47
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τ3(ns) τav(ns)
τav, represent the average lifetime. τ1, τ2, τ3 and a1, a2, a3 represent the exponential fitting life time values and their contribution to average lifetime respectively.
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value of the unbound probe in the same solvent system (2.75 ns) illustrating the fact that NBD recovers its original fluorescence that was quenched due to aggregation on silica surface. To evaluate the response time required for fluoride ion detection in a sample, we have performed a time-dependent steady state study of the variation in fluorescence intensity of the system, at 5x10-5 M fluoride ion concentrations in 1mg/ml solution of NBDAP-MCM (Figure 8). The fluorescence of the reaction mixture has been measured at various intervals of time, ranging from 0 to 380 minutes. During the investigation, a continuous enhancement in the sensor fluorescence has been observed (Figure 8). As can be seen from the curve that nearly 3 fold fluorescence increases is observed in the first 40 minutes of reaction, which is sufficient to signal the presence of fluoride in a solution, hence, the response time of the system can be assigned to be as low as 40 minutes. It is evident from the constant increment of fluorescence, that fluoride is gradually able to diffuse through the mesoporous material, resulting in more NBD moieties being liberated from the solid surface, and add to system's fluorescence. The fluorescence enhancement saturates at about 240 minutes after fluoride addition, at which, the fluorescence has enhanced by nearly 6-7 fold of the initial value.
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Fig 8. Time dependent photoluminescence spectra of NBD-AP-MCM -5 particles (1mg/ml) upon attack by 5x10 M tetrabutylammonium fluoride, in acetonitrile: water (7:3) solvent (pH=2.5), from 0 minutes (black) to 380 minutes. Inset shows a plot of emission intensity as a function of time (λexc. = 460 nm).
Fig 10. Photoluminescence intensity of NBD-AP-MCM particles (1mg/ml), -4 upon attack by 1x10 M tetrabutylammonium fluoride, in acetonitrile: water (7:3) solvent, as a function of pH of the solution (λ exc. = 460 nm, λemi. = 541 nm). 30
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Fig 9. Change in fluorescence enhancement of NBD-AP-MCM (1mg/ml) -5 system upon addition of 5x10 M of tetrabutylammonium salt of various anions in acetonitrile: water (7:3) solvent (pH=2.5). Inset shows the emission plots corresponding to the bar graph (λexc. = 460 nm).
It may be noted here that the system is able to exhibit high fluorescence intensities even for low concentration of fluoride if sufficient time is given. As a result, the observation also gives us a clue to how to fine tune the sensitivity of the sensor system in terms of response time. Now, it can be easily inferred from the above study on the mechanism of fluoride signalling by NBD-AP-MCM, that the event of fluoride detection is a result of a specific reaction, particular to fluoride ion only, and hence the probe is expected to show high selectivity towards fluoride ion. Fluoride is known to have very high affinity to attack Si-O bonds, compared to other anions2,4-7. Fluoride is the most electronegative of all elements, and it has the least size among all comparable anions. Hence the
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effect produced by a chemical moiety with such a unique charge density, cannot be replicated by other anions. This is the key factor which enables the sensor to distinctively identify the fluoride anion. Selectivity of NBD-AP-MCM towards fluoride has been assayed against chloride, bromide, iodide, acetate and bisulphate anions using 5x10-5 M of each ion, in 1mg/ml suspension of NBD-AP-MCM and stirring for two hours, to determine the effect of these competing anions on the sensor fluorescence (Figure 9). As expected, the present sensor system have exhibited a remarkable discrimination between fluoride and other ions, displaying a considerable fluorescence enhancement upon fluoride addition, whereas an insubstantial change in fluorescence has been observed in the cases of other anions (Figure 9). The fluoride sensing experiments reported in this paper, are conducted at pH = 2.5, to maximize the sensitivity of fluoride detection. We have also investigated the fluorescence response of the system in absence and presence of fluoride at various other pH. In absence of fluoride fluorescence response of the present systems is observed to be not affected significantly (see supplementary material). However, a significant change has been observed while monitoring the signalling event of the system in presence of fluoride (Figure 10). From the figure, it is clear that 2.5 to 3.5 is the range of pH, where fluoride signalling event takes place with maximum efficiency. The observation is consistent with literature report where Si-O bond cleavage has been observed at low pH (~2.5). 20We would like also like mention that in pure water the change (fold) in fluorescence enhancement has been measured to very low (1.2-1.5), with no noticeable change in lifetime. This observation is not surprising when we take into account the fact that, both fluorescence quantum yield and lifetime of NBD systems are observed to be very low in pure water.25,33 The increase in non-radiative rate constants due to hydrogen bonding interaction between the NBDbased system and water is primarily responsible for low fluorescence yield and excited state lifetime of the system. 25,33 However, upon increasing the concentration of organic solvent (acetonitrile), in the medium to 7:3 (acetonitrile : water, v/v), the lifetime significantly increased to 2.7 ns. Cellular uptake studies reveal that the system is not toxic to cells and can be used in cellular imaging purposes (see supplementary material).
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5. Conclusion
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A 7-nitro-2,1,3-benzoxadiazole (NBD) anchored mesoporous silica system, NBD-AP-MCM, was synthesized, and characterized using powder-XRD, FE-SEM, EDX, FTIR and Thermogravimetric analysis. The system was shown to sensitively and selectively discriminate fluoride in an aqueous/organic (3:7, pH = 2.5) solvent system. Initially present in completely quenched state, due to aggregation caused quenching (ACQ) of fluorophore, the system displayed up to six fold fluorescence enhancement upon fluoride attack, which is attributed to a specific Si-O bond cleavage reaction, leading to transfer of fluorophore to the organic/aqueous solvent system and subsequent regaining of fluorescence. The working range of concentration for fluoride sensing was estimated to be 10 -5 M to 10-3 M, and the response time can be as low as 40 minutes. The workable pH conditions are pH = 2.3 to 3.5 for sensitive measurement. Further steady state and time dependent studies have been carried out to confirm and explore the mechanistic details of transition of fluorophore from solid to solution phase. Additionally, cellular uptake studies are conducted (see supplementary material) to evaluate the system's biocompatibility, and its application in cellular imaging studies.
Acknowledgement 25
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M.S. thanks the National Institute of Science Education and Research (NISER), Bhubaneswar for funding. G.J. is thankful to the Department of Science and Technology (DST), New Delhi for providing the Kishore Vaigyanik Protsahan Yojana (KVPY) fellowship. A.N. is thankful to Council of Scientific and Industrial Research (CSIR), New Delhi, for the fellowship awarded to him.
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Notes and references a
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School of Chemical Sciences, National Institute of Science Education and Research, Bhubaneswar 751005, India. Fax: +91-674-2302436 Tel: +91-674-2304037 Email: [email protected] b School of biological Sciences, National Institute of Science Education and Research, Bhubaneswar 751005, India 1. L. H. Weinstein, A. Davison, Fluorides in the environment: effects on plants and animals, CABI, 2003. 2. Y. Zhou, J.F. Zhang, J. Yoon, Chem. Rev. 2014, 114, 5511. 3. C. R. Wade, A. E. J. Broomsgrove, S. Aldridge, F. P. Gabbaï, Chem. Rev. 2010, 110, 3958. 4. X. Li, X. Gao, W. Shi, H. Ma, Chem. Rev. 2014, 114, 590. 5. T. H. Kim, T. M. Swager, Angew. Chem. Int. Ed. 2003, 42. 4803. 6. P. Sokkalingam, C. H. Lee, J. Org. Chem. 2011, 76, 3820. 7. R. Hu, J. Feng, D. Hu, S. Wang, S. Li, Y. Li, G. A. Yang, Angew. Chem. Int. Ed. 2010, 49, 4915. 8. D. E. Gómez, L. Fabbrizzi, M. Licchelli, J. Org. Chem. 2005, 70, 5717. 9. J. Shao, H. Lin, H. K. Lin, Talanta 2008, 75, 1015. 10. S. Rochat, K. Severin, Chem. Commun. 2011, 47, 4391. 11. P. Thiampanya, N. Muangsin, B. Pulpoka, Org. Lett. 2012, 14, 4050. 12. T. Agou, M. Sekine, J. Kobayashi, T. Kawashima, Chem. Commun. 2009, 1894. 13. S. V. Bhosale, M. B. Kalyankar, S. J. Langford, Org. Lett. 2009, 11, 5418. 14. O. M. Cabarcos, C. J. Weinheimer, J. M. Lisy, S. S. Xantheas, J. Chem. Phys. 1999, 110, 5 15. S. Y. Kim, J. I. Hong, Org. Lett. 2007, 9, 3109-3112. 16. S. Y. Kim, J. Park, M. Koh, S. B. Park, J. I. Hong, Chem. Commun. 2009, 4735.
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17. L. B. Desmonts, D. N. Reinhoudt, M. C. Calama, Chem. Soc. Rev. 2007, 36, 993. 18. B. J. Scott, G. Wirnsberger, G. D. Stucky, Chem. Mater. 2001, 13, 3140. 19. J. L. Shi, Z. L. Hua, L. X. Zhang, J. Mater. Chem. 2004, 14, 795. 20. A. B. Descalzo, D. Jiménez, J. E. Haskouri, D. Beltrán, P. Amorós, M. D. Marcos, R. Martínez-Máñez, J. Soto, Chem. Commun. 2002, 562. 21. H. Meng, M. Xue, T. Xia, Y. L. Zhao, F. Tamanoi, J. F. Stoddart, J. I. Zink, A. E. Nel, J. Am. Chem. Soc. 2010, 132, 12690. 22. M. Vallet-Regí, F. Balas, D. Arcos, Angew. Chem. Int. Ed. 2007, 46, 7548. 23. I. I. Slowing, J. L. Vivero-Escoto, C. W. Wu, V. S. Y. Lin, Adv. Drug Delivery Rev. 2008, 60, 1278. 24. X. Xinjian Yang, Z. Li, M. Li, J. Ren, X. Qu, Chem. Eur. J. 2013, 19, 15378. 25. S. Saha, A. Samanta, J. Phys. Chem. A. 1998, 102, 7903. 26. A. J. Boulton, P. B. Ghosh, A. R. Katritzky, J. Chem. Soc. B, 1966, 1004. 27. H. Ritter, M. Nieminen, M. Karppinen, D. Brühwiler, D. Microporous and Mesoporous Mater. 2009, 121, 79. 28. S. M. Alahmadi, S. Mohamad, M. J. Maah, Adv. Mat. Sci. Eng. 2013, 8. 29. B. Marler, U. Oberhagemann, S. Vortmann, H. Gies, Microporous Mater. 1996, 6, 375. 30. F. J. Trindade, J. F. Q. Rey, S. Brochsztain, Dyes and Pigments. 2011, 89, 97. 31. A. Chattopadhyay, Chem. Phys. Lipids 1990, 53, 1. 32. S. Mazeres, V. Schram, J. F. Tocanne, A. Lopez, Biophys. J. 1996, 71, 327. 33. S. Lin, W. Struve, Photochem. Photobio. 1991, 54, 361. 34. I. Candel, P. Calero, R. Martínez-Máñez, F. Sancenón, M. D. Marcos, T. Pardo, J. Soto, Inorg. Chim. Acta 2012, 381, 184-194. 35. M. Soni, S. K. Das, P. K. Sahu, U. P. Kar, A. Rahaman, M. Sarkar, J. Phys. Chem. C, 2013, 117 (27),14338. 36. D. Majhi, S. K. Das, P. K. Sahu, S. M. Pratik, A. Kumar, M. Sarkar, Phys. Chem .Chem. Phys. 2014, 16, 18349. 37. C. T. Chen, Chem. Mater. 2004, 16, 4389. 38. F. J. Trindade, J. F. Q. Rey, S. Brochsztain, J. Colloid Interface Sci. 2012, 368, 34. 39. A. Dreuw, J. Plötner, L. Lorenz, J. Wachtveit, J. E. Djanhan, J. Brning, T. Metz, M. Bolte, M. Schmidt, Angew. Chem. Int. Ed. 2005, 44, 7783. 40. B. M. Aveline, T. Hasan, R. W. Redmond, J. Photochem. Photobiol. B 1995, 30, 161. 41. W. Z. Yuan, P. Lu, S. Chen, J. W. Y. Lam, Z. Wang, Y. Liu, H. S. Kwok, Y. Ma, B. Z. Tang, Adv. Mater. 2010, 22, 2159. 42. A. K. Bhoi, S. K. Das, D. Majhi, P. K. Sahu, A. Nijamudheen, N. Anoop, A. Rahaman, M. Sarkar, J. Phys. Chem. B 2014, 118, 9926. 43. S. K. Das, S. S. Misra, P. K. Sahu, A. Nijamudheen, V. Mohan, M. Sarkar, Chem. Phys. Lett. 2012, 546, 90. 44. V. Zaikin, J. M. Halket, A Handbook of Derivatives for Mass Spectrometry, IM Publications, 2009.
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