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Highly fluorescent sensing of nitroaromatic explosives in aqueous media using pyrene-linked PBEMA microspheres
Author’s Accepted Manuscript Highly Fluorescent Sensing of Nitroaromatic Explosives in Aqueous Media using Pyrene-Linked PBEMA Microspheres Hamza Turhan, Ece Tukenmez, Karagoz, Niyazi Bicak
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S0039-9140(17)31110-4 https://doi.org/10.1016/j.talanta.2017.10.061 TAL18059
To appear in: Talanta Received date: 6 July 2017 Revised date: 24 October 2017 Accepted date: 28 October 2017 Cite this article as: Hamza Turhan, Ece Tukenmez, Bunyamin Karagoz and Niyazi Bicak, Highly Fluorescent Sensing of Nitroaromatic Explosives in Aqueous Media using Pyrene-Linked PBEMA Microspheres, Talanta, https://doi.org/10.1016/j.talanta.2017.10.061 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.
Highly Fluorescent Sensing of Nitroaromatic Explosives in Aqueous Media using Pyrene-Linked PBEMA Microspheres Hamza Turhan, Ece Tukenmez, Bunyamin Karagoz*, Niyazi Bicak* Istanbul Technical University, Department of Chemistry, Maslak, 34469 Istanbul, Turkey *
Corresponding authors:
Assoc. Prof. Bunyamin Karagoz Istanbul Technical University, Department of Chemistry, Maslak, 34469 Istanbul, Turkey Tel:(+90 212 285 3261); Fax:(+90 212 285 6386) Email: [email protected]
Prof. Niyazi Bicak Istanbul Technical University, Department of Chemistry, Maslak, 34469 Istanbul, Turkey Tel:(+90 212 285 3261); Fax:(+90 212 285 6386) Email: [email protected]
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ABSTRACT Crosslinked 2-bromoethyl methacrylate polymer (PBEMA) was prepared in micro-spherical form (2- 5 µm) by precipitation polymerization methodology. The bromide substituent was substituted
with
an
azide
Propynyloxy)methyl]pyrene]
via
group,
which
alkyne-azide
was click
then
coupled
chemistry.
The
with
1-[(2-
pyrene-linked
microspheres showed an intense green-blue excimer emission with a maximum at 480 nm, implying π−π stacking between the pyrene moieties on the microsphere surfaces. This fluorescence emission is extremely sensitive to the aromatic nitro compounds. So that the green-blue light fades immediately upon addition of trace amounts of 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (DNT) and 2,4,6-trinitro phenol (TNP) in 100 % aqueous media. Stern-Volmer plots were employed for comparison of their fluorescence quenching effects. The plots revealed Stern-Volmer constants of 1.33×105, 2.451×105 and 1.076×105 M-1 for TNT, TNP and DNT, respectively. Furthermore, it has been observed that, the microspheres can be reused several times, without losing excimer emission properties. Keywords: 2-Bromoethyl methacrylate (BEMA), pyrene, click chemistry, nitroaromatic detection, fluorescence quenching, aqueous phase. 1.Introduction Detection of nitroaromatic compounds with high accuracy are essential and have a vital importance due to their antiterrorism and environmental applications [1,2]. As known, nitroaromatic compounds especially TNT, DNT and TNP are the most commonly used explosives components in defense industry [3,4]. Besides the devastating impact, these environment pollutant compounds have extremely toxic effects on living organisms [5]. Nitroaromatic compounds, for instance TNP, lead to severe health effects such as strong irritation to the skin-eye and severe respiratory disorders [6]. Owing to this significant toxicity TNT, DNT and TNP have been included in the Environmental Protection Agency List of Priority pollutants [7]. To date various significant and sophisticated methods such as gas chromatography with a mass spectrometer [8], nuclear quadrupole resonance [9], X-ray diffraction [10], enhanced Raman spectrometry [11], have been employed for the detection of those materials. However, these techniques are not very applicable for detection of explosives
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in the field because of high cost, complexity, low precision and time consuming nature of the manipulations [12,13]. Compared to the other methods, sensing via fluorescence manipulation approach has attracted great attention due to introducing many different peculiarities such as low-cost, high sensitivity, quick response, simplicity of detection of these pollutant compounds [14-16]. In recent years, for this purpose, various materials such as fluorescent conjugated polymers [17,18] and fluorescent small molecules [19,20], nanofibers [21,22], nanoparticles [23,24], metal−organic frameworks [25-27] and micro/nanoaggregates [28] have been designed. Furthermore, most of these fluorescent materials can be easily incorporated into microelectronic devices with a very low-cost [29]. Briefly, detection of the nitro compounds relies on fluorescence manipulation [30], in most cases, fluorescence quenching takes place during the interaction of the fluorescent material with electron deficient energetic materials via photo induced electron transfer from excited fluorescent molecules to analytes [31]. Despite all the detection peculiarities of these fluorescent based materials, most of them are unfortunately in hydrophobic nature [32,33] and this incompatibility brings new problems in aqueous medium detection such as limited selectivity, low sensitivity, on-site detection incapability and unviability [34,35]. Development of fluorescent material with ultrasensitivity and high selectivity in 100 % aqueous medium and solid state for detection of energetic materials desirable and necessary field of research [36-38]. Among the fluorescent probes, pyrene-based ones are widely used as efficient fluorophores due to their high quantum yield, and chemical stability on molecular labeling and high fluorescent sensing behaviors [39-41]. In this respect, Bayindir and co-workers successfully demonstrated porous ormosil thin film having almost constant stable bright pyrene excimer emission [42]. In this study, the pyrene excimer moiety rapidly quenched in the presence of nitroaromatic compounds based on electron-transfer between π−π* stacked pyrene molecules and nitroaromatic molecules [43]. Fang group also investigated sensing nitroaromatic compounds via pyrene containing conjugated polymer based thin film on glass substrate [44,45]. Both studies revealed that pyrene derivatives with high excimer emission occurrence are very effective materials for sensing nitroaromatic compounds. Another paper exists in the literature discussing the use of a polymeric fluorescent material. They reported polystyrene microspheres with pyrene acrylate and perylene bisimide with diacrylate functions yielding tunable colors and sensing material for detection of 4-nitrotoluene in vapor phase which is quite different from our approach [46].
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In this report, detection ability of highly fluorescent the pyrene-linked and crosslinked PBEMA (poly 2-bromoethyl methacrylate) microspheres was investigated on nitroaromatic compounds. For this purpose, highly uniform bromide having the crosslinked PBEMA microspheres (2-5 µm) were synthesized in the presence of AIBN (2,2’-azo bis(isobutyronitrile)) via precipitation polymerization. Bromide groups on the microspheres were then converted to the azide groups by a simple substitution using sodium azide (NaN3). In the last step, 1-[(2-Propynyloxy)methyl]pyrene] units were anchored on the microspheres by azide-alkyne click chemistry [47-49]. The resulting promising fluorescent material was employed for the detection of nitroaromatic compounds. As a high excimer formation of the pyrene functional PBEMA microbeads in 100 % aqueous media makes very sensitive to nitroaromatics.
2. Materials and methods 2.1. Instrumentation FT-IR spectra were recorded on a Perkin Elmer FT-IR Spectrum One B spectrometer. UV spectra were recorded on a Shimadzu UV-1601 spectrophotometer (Supporting Information, Figure S7). The fluorescence lifetime measurements were carried out by timeresolved spectrophotometer technique using “PTI C-71 Time Master”. Aqueous stock solution (0.05mg/mL) of PBEMA microbeads were used for the measurements. Fluorescence measurements were carried out at room temperature using Agilent Cary Eclipse fluorescence spectrophotometer using conventional cell with 1 cm path length. The slit-width for the fluorescence experiment was kept constant at 5 nm (excitation) and 5 nm (emission) and the excitation wavelength was set at 342 nm. Particle shapes and sizes of the product were investigated using scanning electron microscopy (SEM, FEI-Quanta 200 FEG). Prior to taking SEM images, microsphere samples were placed on metal stubs by using double-sided copper tape, and in order to minimize the charging problem during SEM examination, samples were sputtered with 5 nm of Au/Pd (PECS-682). Fluorescence image of the microscope were obtained using an Olympus Bx60 Transmitted-Reflected Light Microscope equipped with a Nikon digital camera. It has UplanFL 10×/0.30, UplanFL 20×/0.50, UplanFL 40×/0.75, UplanFL 100 microspheres ×/1.3 oil objectives and all objectives have DIC optics. In order to take light microscopy images, the microspheres were dispersed in water, and applied onto glass microscope slides.
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2.2. Materials 2-Bromoethyl methacrylate was synthesized as reported before explained in the literature [50]. Propargyl bromide (Sigma-Aldrich, 98 %) and 1-Pyrenemethanol (SigmaAldrich, 98 %) were used as received without any further purification. Acetonitrile (SigmaAldrich, 99 %,) was redistilled over CaH2 before use. 2,2’-azo bis-(isobutyronitrile) (AIBN, Fluka) was recrystallized from methanol. Methyl methacrylate (MMA, Sigma-Aldrich) and ethylene glycol dimethacrylate (EGDMA, Sigma-Aldrich) were passed through a column containing basic alumina before use to remove the inhibitors. Water used throughout the experiments was deionized first and then distilled twice. 2,4,6-trinitrotoluene (TNT) (1.00 mg/mL in acetonitrile), 2,4-dinitrotoluene (DNT) (5000 µg/mL in methanol), 2,4,6trinitrophenol (TNP) in water, 4-nitrotoluene (4-NT), nitromethane (NM), nitrophenol (NP) and nitrobenzene (NB) are of analytical grade chemicals purchased from Sigma Aldrich and used directly without further purification. All the other chemicals were used as purchased.
2.3. Preparation of the PBEMA microspheres via precipitation polymerization In this recipe, BEMA (as monomer, 2.89 g or 15 mmol), EGDMA (as crosslinker, 6.94 g or 35 mmol) and AIBN (0.24 g, 1.46 mmol - 2 wt %) were mixed in 360 mL acetonitrile in a three-necked flask (500 mL) under nitrogen atmosphere. The flask was placed in a thermostated oil-bath and equipped with a mechanical stirrer under inert atmosphere, and a nitrogen gas streaming was provided for maintaining a nitrogen atmosphere. Then, polymerization reaction started and carried out at 70 oC under continuous stirring (~30 rpm) for 24 h. Thereafter, the reaction content was cooled to the room temperature and filtered off. Resulting crosslinked beads were washed successively with tetrahydrofuran (THF) (40 mL x 2), methanol (40 mL x 2) and diethyl ether (30 mL). The product was kept under vacuum at 40 oC overnight. The yield of the product was calculated by gravimetrically and found at around 54 %.
2.4. Determination of bromide content of the PBEMA microbeads Bromide content of the microbeads was found gravimetrically by silver bromide (AgBr) precipitation method [51]. In this procedure 0,4 g of the PBEMA microsphere was treated with 20 mL of methanolic NaOH solution (20 wt %) and refluxed for 24 h in the presence of a reflux condenser. Then the reaction mixture was filtered off with blue band filter paper and washed with distilled water (20 mL). All of the filtrate were collected in a 50 5
mL volumetric flask and the needed amount was completed with distilled water. Half of this filtrate was neutralized with nitric acid and mixed with excess amount of AgNO3 solution (1 M). The resulting AgBr precipitate was filtered off, washed with water and dried at 40 oC under atmospheric pressure. The AgBr was weighted as 0.057 ± 0.002 g and this indicated 0.76 mmol.g-1 of accessible bromide content.
2.5. Azidation of the PBEMA microspheres The bromide group on the PBEMA microbeads was substituted with azide ones by reaction with NaN3. For this purpose, the PBEMA microsphere (1 g) was mixed with NaN3 (0.325 g, 5 mmol) in 25 mL dimethylformamide (DMF) with a small amount of distilled water to enhance the solubility of sodium azide. The reaction mixture was sealed and covered with aluminum foil to avoid light exposure and shaken for 24 h at room temperature. The resulting PBEMA bead product was isolated by filtration and successive washings with water (30 mL × 2), methanol (30 mL) and dried overnight under vacuum at room temperature.
2.6. Synthesis of 1-[(2-Propynyloxy)methyl]pyrene](POMP) The propargyl pyrene was synthesized according to previous report [49]. 1- Pyrene methanol (1.16 g, 5 mmol) was reacted with sodium hydride (60 wt % dispersion in oil) (0.131 g, 5.5 mmol) in the presence of 20 mL dry THF and the reaction mixture was stirred at 0 oC under nitrogen for 1 h. Then propargyl bromide (0.654 g, 5.5 mmol) in toluene was added dropwise to the solution and the reaction carried out for 24 h at room temperature. After then the mixture was refluxed for 4 h in the dark. The reaction solution was extracted with ethyl acetate and half of the solvent evaporated. A light yellow product was obtained after the solvent removal. The raw product was dissolved in toluene and passed through the basic alumina column to remove unreacted 1-pyrene methanol. After removing toluene by evaporation, the residue was dried under vacuum oven (Yield: 58 %). 1H NMR (500 MHz, CDCl3) δ:8.00-8.42 (m, 9H, Ar H), 5.35 (s, 2H, CH2), 4.34 (d, 2H, CH2).
2.7. Preparation of the pyrene-linked PBEMA microspheres via click reaction The crosslinked PBEMA microspheres (0.2 g, containing 0.15 mmol azide groups) were dispersed in 4 mL DMF in a flask. 1-[(2-Propynyloxy)methyl]pyrene] (77 mg, 0.3 mmol), CuBr (22.8 mg, 0.15 mmol) and PMDETA (33.3 μL, 0.15 mmol) were then added to the mixture under inert atmosphere. The reaction was carried out at 40 oC with continuous 6
stirring for 48 h. The resulting pyrene-linked PBEMA microspheres were separated from the mixture by centrifugation. For the purification purpose, the resulting crude solid microbeads at room temperature was re-dispersed in DMF and separated by centrifugation. This purification step was repeated three times with dilute acidic methanol solution and THF. After purification, the resulting microbeads were dried overnight under vacuum and weighted as 215 mg.
2.8. Fluorescence detection procedure The PBEMA microsphere stock solution was prepared by dispersing 5 mg of microsphere in 100 mL of pure water. The suspension was sonicated for 1 hour. 4.4x10-4, 5.5x10-4 and 6x10-4 M of the stock solution of nitroaromatic compounds was prepared by adding 10, 10.1, 13.8 mg of TNT, DNT and TNP in 100 mL pure water. In each case, 3 mL of the PBEMA microspheres suspension (0,05mg/mL) was put into a quartz cuvette (1 cm length) with a magnetic stirrer and individually TNT, DNT and TNP aqueous solution (5μL) was added gradually onto the quartz cuvettes by using microliter syringe. The total concentration was changed from 0 to 11 μM. For each addition, at least three fluorescence spectrums were recorded at 25 oC to obtain a concordant value. The λ excitation was chosen 342 nm with 5 nm slit width. Note that the pyrene emissions (excimer at 480 nm, monomer at 396 nm) were quenched upon addition of nitroaromatics.
3.Results and discussion 3.1. Preparation of the pyrene functional crosslinked microbeads This work is aimed for the design and synthesis of dense pyrene-linked microbeads exhibiting an intense fluorescence for sensing and detection of nitro compounds in trace quantities. The synthesis pathway involves, i) Crosslinking copolymerization of 2-bromoethyl methacrylate with ethylene glycol dimethacrylate by the precipitation polymerization to give 2-5 µm size particles. The precipitation polymerization was deliberately chosen to attain high surface area-to-volume ratios. ii) in the second step the bromide substituents were replaced with azide groups. iii) Then, pyrene fluorophore was anchored to the micro-particle surfaces by alkyne-azide click chemistry using 1-[(2-Propynyloxy)methyl]pyrene] as depicted in Scheme 1. The bromide content of the microspheres was estimated to be 0.76 mmol/g by AgBr method. However according to feed composition in the polymerization, the real 7
bromide concentration should be around 5.0 mmol per gram. This implies that only 15.2 % of total bromide is accessible. Remaining portion, 85 % has been embedded in the crosslinked polymer matrix. The accessible portion must be located at the surfaces of the microspheres. This means that, accessible azide groups are located at the surfaces, which are amenable to click with POMP in the last step. The SEM picture also has showed no mechanical crack which implies retaining of the spherical shapes in the surface modifications (Supporting Information, Figure S1). FTIR spectra of the microspheres indicate stepwise transformations in each stage. Thus, FTIR spectrum of the starting microspheres (Fig. 1a) shows typical bromoethyl methacrylate polymer vibrations. The carbonyl and ester group stretching vibrations appear at 1725 and 1070 cm-1 respectively. A weak peak around 610 cm-1 can be ascribed to C-Br vibration. After substituting the bromide function with azide group, a narrow sharp peak emerges at 2105 cm−1 that can be ascribed to occurrence of the substitution reaction (Fig. 1b). This peak becomes almost invisible after clicking with POMP (Fig. 1c) in the last step. And also new absorption peak appeared at 3140 cm-1 implying C-H stretching vibration of aromatic moieties belonging to surface tethered pyrene groups on the microbeads. Fluorescence microscope image of the pyrene-linked microbeads in Fig. 2 (left) shows glistening particles emitting intense fluorescence. Optical microscope image of the product in Fig. 2 (right) shows almost uniform-sized particles in spherical shape with average size of 2-5 µm (Supporting Information, Fig. S2).
3.2 Examination of fluorescence behavior of the pyrene-linked microspheres The fluorescence (PL) properties of pyrene-linked microspheres were investigated using water as the dispersing medium. For comparison of the fluorescence behaviors, first, emission spectrum of 1-[(2-Propynyloxy)methyl]pyrene] was taken in aqueous dispersion. PL emission spectrum of the free 1-[(2-Propynyloxy)methyl]pyrene] exhibits three emission bands at 380, 396 and 420 nm as observed in Fig. 3 (black). UV absorption bands of 8
this compound lie below 350 nm. These peaks are associated with Stocks shift of transitions appeared in its absorption spectrum. Whereas in PL emission spectrum of pyrene functional microspheres (Fig. 3 red) represents two broad bands centered at 396 nm and 480 nm. The strong emission at 480 nm that is assigned as excimer emission. This must be due to close proximity of surface bound pyrene moieties. Such a proximity facilitates ππ interaction of excited pyrene with another pyrene in ground state (excimer formation). In other words, the excimer emission band at 480 nm is observable even by naked eye and originates from luminescence of pyrene-excited pyrene conjugates. Therefore, organic compounds that are able to destroy this conjugate can quench excimer emission. Additionally, we have also investigated fluorescence lifetime of the pyrene tethered microbeads. For this purpose, 0.05mg/mL stock solution of the particles were used in this experiment. Results indicate, decaying one with 11.84 ±1.7 nanoseconds (ns) of lifetime and another one with 49.87 ± 1.17 ns of lifetime. Apparently, the longer lifetime represents the pyrene attached to inner part of the particle and the fast one shows emission of the pyrene located at the surfaces. Presumably this is due to easy energy transfer from excited outmost pyrenes to the solvent molecules. Stability of the pyrene linkage on the microbeads was also checked by simply monitoring of the excimer emission intensity in the presence and absence of the light. During the daylight interaction, fluorescence plot of the microbeads show decrease on the excimer emission band at around 480 nm due to the photo-decomposition of the ether linkages via light induced peroxide formation. In the same plot, an increment of free pyrene emission band were observed day by day due to the cleavage, as expected. Whereas, the samples stored in the dark did not show any noticeable decrease of PL emission. The results were collected in Fig. S9. In this work, we have investigated quenching effect of some aromatic nitro compounds on the green-blue excimer emission of surface-bound pyrene. This was achieved simply by adding 33 µL from stock solutions (1mM) of the TNT, DNT and TNP individually onto 3 mL aqueous dispersions of pyrene functional microspheres (0.05 mg/mL) while fluorescing. An immediate fading of the excimer emission was observed in all cases as depicted in Fig. S3 (see supporting information). This result clearly indicates high sensitivity of the pyrene-linked microspheres towards nitroaromatics. Quenching efficiencies (QE) of the nitro compounds in this condition were determined 86.7 %, 92.2 %, 85 % for TNT, TNP and DNT respectively. The quenching efficiencies (QE) were estimated based on the following relationship, 9
Io
100
where I0 and I are microsphere excimer fluorescence intensities at 480 nm in the absence and presence of nitroaromatics. QE of 2,4,6-trinitro phenol is somewhat higher than those of the others. Considering π electron densities, TNP having the lowest electron deficiency is expected to induce low or moderate extinction effect on the excimer emission. This controversy can be explained in terms of acid-base interaction between acidic OH group of 2,4,6-trinitro phenol and basic triazole connecting group. Three nitro groups on the phenol moiety make it much more acidic, so that TNP tends to form salt with triazole ring and this results in quick distraction of the excimer with 2,4,6-trinitro phenol.
3.3. Quenching of the excimer emission as a function of nitroaromatic concentrations Logically quenching efficiency of nitroaromatics must be concentration depended. To investigate effects of the quencher concentrations a series of experiments were performed, in which the stock solutions of TNT, TNP and DNT were gradually added into pyrene-linked microsphere dispersions (0.05 mg/mL) using microliter syringes. In these experiments, the final concentrations were maintained in 0-50 µM range and the excimer emission intensities were measured. The relevant quenching efficiencies derived from these data were plotted against the concentrations of nitroaromatics, as depicted in Fig. 4. The column graphs in this figure revealed that increasing concentrations increase
’s,
however there is no linear relationship. The highest QE is attained by TNP and reaches to 92.2 for the concentration of 50 µM as discussed above. Further fluorescence behavior of pyrene functional microspheres was explored in the presence of various nitro compound 4nitrotoluene (4-NT), nitromethane (NM), nitrophenol (NP) and nitrobenzene (NB). It can be seen that there was no significant effect on fluorescence intensity. The results suggest that the fluorescent microspheres are highly selective for explosive detection. In order to inspect validity of Stern-Volmer quenching mechanism, the data collected were employed to construct Stern-Volmer plots appeared in the windows of Fig. 5. SternVolmer equation is given as, [52] I0 /I=1+Ksv [Q] 10
where I0 and I are fluorescence intensities in the absence and presence of a quencher and [Q] represents the quencher concentration. Ksv denotes Stern-Volmer constant which depends on solvent and nature of the quencher and fluorescence source. Linearity of I0 /I versus [Q] plots implies validity of the Stern-Volmer quenching mechanism for the nitroaromatics studied. The regression factors (R2) were obtained as 0.991, 0.981, and 0.988 for TNT, TNP, and DNT respectively. Closeness of the regression factors to unity indicates good linearity in all cases. Stern-Volmer constants obtained from the slopes of curves were determined the 1.33×105, 2.451×105 and 1.076×105 M-1 for TNT, TNP, and DNT respectively. Those high Ksv values imply possibility of precise estimation of nitroaromatics by this system. The highest Ksv for the case of 2,4,6-trinitro phenol indicates its extreme sensitivity in quenching of the excimer emission. The detection limits of TNT, TNP and DNT have been determined to be 2.1×10-7 M, 1.14×10-7 M and 2.6×10-7 M respectively, one of the most sensitive sensors that have been reported and used in aqueous solution (Supporting information, Table S1). We also demonstrated a simple solid phase detection of TNT and TNP via adding different concentration on microspheres adsorbed TLC plate (Shown in Fig.S5 and Fig.S6 in Supplementary Material).
3.4. The solvent effect Solvent effect is known as an important parameter for both the fluorescence intensity and quenching. Excimer formation must also be dependent on solvent type. According to the literature there is a strong relationship between the excimer formation and the solvent polarity [53]. To investigate the solvent effect in this work, the fluorescence spectra of pyrene-linked microspheres were recorded while contacting of the microspheres with different solvents. All the spectra were collected in Fig. 6. According to Fig. 6, the intensities of all the emission bands are solvent dependent. Being non-polar n-hexane give emission bands with lowest intensities. Polar solvents are expected to induce formation of perfectly overlapped sandwich type excimer. Herein the solvent polarities are in order of water > acetonitrile > methanol > THF > hexane, whereas excimer/monomer (Iex / Imon) ratio is in order of water > methanol > acetonitrile > THF > hexane. Although excimer emission is stronger in polar solvent there is no order between solvent polarity and excimer intensity according to these results.
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In the case of THF and acetonitrile, intensity of the emission band at 396 nm (monomer emission) is greater than that of the excimer emission. This could be explained by good solvent behavior of THF and acetonitrile on both the pyrene moieties and the naked PBEMA microbeads. After the solvents interaction with the microbeads, swollen microbeads may cause the distance increment between the anchored pyrene groups on the surface of the beads and decreasing of the excimer formation. In the methanol and water cases, the intensity of the excimer emission band is significantly higher than the monomer emission band. These two solvents are not good solvent for both pyrene groups tethered on the surface of the beads and the naked PBEMA beads. So the distance decreases between the pyrene moieties due to the shrinkage of the PBEMA beads and this cause to increase excimer formation.
3.5. Reversibility of the quenching process The effective sensing properties of microsphere were also supported by regeneration of the immobilized pyrenes. TNT was the only quencher used in the regeneration experiments. For this purpose, a UV cell was filled with 3 mL of the microsphere dispersion and aqueous TNT solution (10 μM) was added to the dispersion and the excimer emission was measured. The microspheres were separated by centrifugation and dried at atmospheric pressure at 50 oC for overnight. Being very soluble in acetone, TNT residue was removed by washing the microspheres with 10 mL of acetone twice and dried. Then the excimer intensity was measured once again. The microspheres were recovered by washing with acetone and reused as excimer emission source in the second time. These cycles were repeated for many times and the intensities of the excimer emission were noted. The excimer band intensities were plotted against cycling numbers as seen in Fig. S8. The figure reveals almost constant excimer emission intensity throughout the regeneration cycles. Slight decrease of the excimer emission intensity might be due to mechanical loss of the particles. In another word, it is clear that this sensing process is reversible and recovered particles show good retention of the sensitivity towards aromatic nitro compounds. Reversibility was also observed in solid phase visually (Supplementary Material, Fig.S4).
4. Conclusions Pyrene tethered crosslinked microspheres were prepared by anchoring of 1-[(2Propynyloxy)methyl]pyrene] onto the surface of the microspheres via alkyne-azide click 12
chemistry. Surface bound pyrene moieties tend to form excimer in methanol, water and acetonitrile. This fact stimulates fluorescing a strong and green-blue excimer emission with a maximum at 480 nm. The excimer emission was determined to be very sensitive to TNT, TNP and DNT with very low limit of detection 2,1×10-7 M, 1,14×10-7 M and 2,6×10-7 M respectively. The nitroaromatic contaminants on microspheres can be removed by simple washing with acetone and the resulting particles are reusable many times for precise detection of these nitro compounds. Having these peculiarities, the presented system provides excellent platform for easy detection of explosives even in trace quantities.
Acknowledgements The authors are thankful to Istanbul Technical University graduate school of science engineering and technology for the financial support. We are also grateful to Prof. Dr. Ismail Yilmaz for his helps in taking fluorescence spectra. We are also thankful to YTU Department of Chemistry for fluorescence lifetime measurement.
Appendix A. Supporting Information Various type of microscope images, UV-Vis absorbance spectra and solid state visual detection studies are associated with this article given on supplementary material.
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Figure Captions Fig. 1. FTIR spectra of a) 2-Bromoethyl methacrylate polymer (PBEMA) b) the azide functionalized PBEMA c) the pyrene functionalized PBEMA. Fig. 2. Fluorescence microscope image of the pyrene-linked microspheres showing uniformly distributed size and fluorescence intensities. Fig. 3. Fluorescence emission spectra of free (Black) 1-[(2-Propynyloxy)methyl]pyrene] (2x10-6 M) and the pyrene-linked PBEMA microspheres (Red) (0,05 mg/mL) in water. Fig. 4. Quenching percentage of pyrene functional microspheres for various nitro compounds. (The concentrations varied 0-50 μM) range. Pyrene-linked micro-particles 0.05 mg/mL. λex =342 nm, ex/em slit widths = 5/5 nm). Fig. 5. Full PL spectra of the pyrene-linked microspheres upon the addition of TNT, TNP, and DNT in 100 % aqueous solution (λex 342 nm). The inset is its corresponding SternVolmer plot. Fig. 6. Fluorescence emission spectra of the pyrene anchored microspheres in the presence of different solvents.
Scheme Scheme 1. Preparation of highly fluorescent the pyrene-linked polymer starting from densely crosslinked 2-bromoethyl methacrylate polymer, for detection of nitroaromatics in trace quantities and their SEM images.
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Bunyamin www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(17)31110-4 https://doi.org/10.1016/j.talanta.2017.10.061 TAL18059
To appear in: Talanta Received date: 6 July 2017 Revised date: 24 October 2017 Accepted date: 28 October 2017 Cite this article as: Hamza Turhan, Ece Tukenmez, Bunyamin Karagoz and Niyazi Bicak, Highly Fluorescent Sensing of Nitroaromatic Explosives in Aqueous Media using Pyrene-Linked PBEMA Microspheres, Talanta, https://doi.org/10.1016/j.talanta.2017.10.061 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.
Highly Fluorescent Sensing of Nitroaromatic Explosives in Aqueous Media using Pyrene-Linked PBEMA Microspheres Hamza Turhan, Ece Tukenmez, Bunyamin Karagoz*, Niyazi Bicak* Istanbul Technical University, Department of Chemistry, Maslak, 34469 Istanbul, Turkey *
Corresponding authors:
Assoc. Prof. Bunyamin Karagoz Istanbul Technical University, Department of Chemistry, Maslak, 34469 Istanbul, Turkey Tel:(+90 212 285 3261); Fax:(+90 212 285 6386) Email: [email protected]
Prof. Niyazi Bicak Istanbul Technical University, Department of Chemistry, Maslak, 34469 Istanbul, Turkey Tel:(+90 212 285 3261); Fax:(+90 212 285 6386) Email: [email protected]
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ABSTRACT Crosslinked 2-bromoethyl methacrylate polymer (PBEMA) was prepared in micro-spherical form (2- 5 µm) by precipitation polymerization methodology. The bromide substituent was substituted
with
an
azide
Propynyloxy)methyl]pyrene]
via
group,
which
alkyne-azide
was click
then
coupled
chemistry.
The
with
1-[(2-
pyrene-linked
microspheres showed an intense green-blue excimer emission with a maximum at 480 nm, implying π−π stacking between the pyrene moieties on the microsphere surfaces. This fluorescence emission is extremely sensitive to the aromatic nitro compounds. So that the green-blue light fades immediately upon addition of trace amounts of 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (DNT) and 2,4,6-trinitro phenol (TNP) in 100 % aqueous media. Stern-Volmer plots were employed for comparison of their fluorescence quenching effects. The plots revealed Stern-Volmer constants of 1.33×105, 2.451×105 and 1.076×105 M-1 for TNT, TNP and DNT, respectively. Furthermore, it has been observed that, the microspheres can be reused several times, without losing excimer emission properties. Keywords: 2-Bromoethyl methacrylate (BEMA), pyrene, click chemistry, nitroaromatic detection, fluorescence quenching, aqueous phase.
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in the field because of high cost, complexity, low precision and time consuming nature of the manipulations [12,13]. Compared to the other methods, sensing via fluorescence manipulation approach has attracted great attention due to introducing many different peculiarities such as low-cost, high sensitivity, quick response, simplicity of detection of these pollutant compounds [14-16]. In recent years, for this purpose, various materials such as fluorescent conjugated polymers [17,18] and fluorescent small molecules [19,20], nanofibers [21,22], nanoparticles [23,24], metal−organic frameworks [25-27] and micro/nanoaggregates [28] have been designed. Furthermore, most of these fluorescent materials can be easily incorporated into microelectronic devices with a very low-cost [29]. Briefly, detection of the nitro compounds relies on fluorescence manipulation [30], in most cases, fluorescence quenching takes place during the interaction of the fluorescent material with electron deficient energetic materials via photo induced electron transfer from excited fluorescent molecules to analytes [31]. Despite all the detection peculiarities of these fluorescent based materials, most of them are unfortunately in hydrophobic nature [32,33] and this incompatibility brings new problems in aqueous medium detection such as limited selectivity, low sensitivity, on-site detection incapability and unviability [34,35]. Development of fluorescent material with ultrasensitivity and high selectivity in 100 % aqueous medium and solid state for detection of energetic materials desirable and necessary field of research [36-38]. Among the fluorescent probes, pyrene-based ones are widely used as efficient fluorophores due to their high quantum yield, and chemical stability on molecular labeling and high fluorescent sensing behaviors [39-41]. In this respect, Bayindir and co-workers successfully demonstrated porous ormosil thin film having almost constant stable bright pyrene excimer emission [42]. In this study, the pyrene excimer moiety rapidly quenched in the presence of nitroaromatic compounds based on electron-transfer between π−π* stacked pyrene molecules and nitroaromatic molecules [43]. Fang group also investigated sensing nitroaromatic compounds via pyrene containing conjugated polymer based thin film on glass substrate [44,45]. Both studies revealed that pyrene derivatives with high excimer emission occurrence are very effective materials for sensing nitroaromatic compounds. Another paper exists in the literature discussing the use of a polymeric fluorescent material. They reported polystyrene microspheres with pyrene acrylate and perylene bisimide with diacrylate functions yielding tunable colors and sensing material for detection of 4-nitrotoluene in vapor phase which is quite different from our approach [46].
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In this report, detection ability of highly fluorescent the pyrene-linked and crosslinked PBEMA (poly 2-bromoethyl methacrylate) microspheres was investigated on nitroaromatic compounds. For this purpose, highly uniform bromide having the crosslinked PBEMA microspheres (2-5 µm) were synthesized in the presence of AIBN (2,2’-azo bis(isobutyronitrile)) via precipitation polymerization. Bromide groups on the microspheres were then converted to the azide groups by a simple substitution using sodium azide (NaN3). In the last step, 1-[(2-Propynyloxy)methyl]pyrene] units were anchored on the microspheres by azide-alkyne click chemistry [47-49]. The resulting promising fluorescent material was employed for the detection of nitroaromatic compounds. As a high excimer formation of the pyrene functional PBEMA microbeads in 100 % aqueous media makes very sensitive to nitroaromatics.
2. Materials and methods 2.1. Instrumentation FT-IR spectra were recorded on a Perkin Elmer FT-IR Spectrum One B spectrometer. UV spectra were recorded on a Shimadzu UV-1601 spectrophotometer (Supporting Information, Figure S7). The fluorescence lifetime measurements were carried out by timeresolved spectrophotometer technique using “PTI C-71 Time Master”. Aqueous stock solution (0.05mg/mL) of PBEMA microbeads were used for the measurements. Fluorescence measurements were carried out at room temperature using Agilent Cary Eclipse fluorescence spectrophotometer using conventional cell with 1 cm path length. The slit-width for the fluorescence experiment was kept constant at 5 nm (excitation) and 5 nm (emission) and the excitation wavelength was set at 342 nm. Particle shapes and sizes of the product were investigated using scanning electron microscopy (SEM, FEI-Quanta 200 FEG). Prior to taking SEM images, microsphere samples were placed on metal stubs by using double-sided copper tape, and in order to minimize the charging problem during SEM examination, samples were sputtered with 5 nm of Au/Pd (PECS-682). Fluorescence image of the microscope were obtained using an Olympus Bx60 Transmitted-Reflected Light Microscope equipped with a Nikon digital camera. It has UplanFL 10×/0.30, UplanFL 20×/0.50, UplanFL 40×/0.75, UplanFL 100 microspheres ×/1.3 oil objectives and all objectives have DIC optics. In order to take light microscopy images, the microspheres were dispersed in water, and applied onto glass microscope slides.
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2.2. Materials 2-Bromoethyl methacrylate was synthesized as reported before explained in the literature [50]. Propargyl bromide (Sigma-Aldrich, 98 %) and 1-Pyrenemethanol (SigmaAldrich, 98 %) were used as received without any further purification. Acetonitrile (SigmaAldrich, 99 %,) was redistilled over CaH2 before use. 2,2’-azo bis-(isobutyronitrile) (AIBN, Fluka) was recrystallized from methanol. Methyl methacrylate (MMA, Sigma-Aldrich) and ethylene glycol dimethacrylate (EGDMA, Sigma-Aldrich) were passed through a column containing basic alumina before use to remove the inhibitors. Water used throughout the experiments was deionized first and then distilled twice. 2,4,6-trinitrotoluene (TNT) (1.00 mg/mL in acetonitrile), 2,4-dinitrotoluene (DNT) (5000 µg/mL in methanol), 2,4,6trinitrophenol (TNP) in water, 4-nitrotoluene (4-NT), nitromethane (NM), nitrophenol (NP) and nitrobenzene (NB) are of analytical grade chemicals purchased from Sigma Aldrich and used directly without further purification. All the other chemicals were used as purchased.
2.3. Preparation of the PBEMA microspheres via precipitation polymerization In this recipe, BEMA (as monomer, 2.89 g or 15 mmol), EGDMA (as crosslinker, 6.94 g or 35 mmol) and AIBN (0.24 g, 1.46 mmol - 2 wt %) were mixed in 360 mL acetonitrile in a three-necked flask (500 mL) under nitrogen atmosphere. The flask was placed in a thermostated oil-bath and equipped with a mechanical stirrer under inert atmosphere, and a nitrogen gas streaming was provided for maintaining a nitrogen atmosphere. Then, polymerization reaction started and carried out at 70 oC under continuous stirring (~30 rpm) for 24 h. Thereafter, the reaction content was cooled to the room temperature and filtered off. Resulting crosslinked beads were washed successively with tetrahydrofuran (THF) (40 mL x 2), methanol (40 mL x 2) and diethyl ether (30 mL). The product was kept under vacuum at 40 oC overnight. The yield of the product was calculated by gravimetrically and found at around 54 %.
2.4. Determination of bromide content of the PBEMA microbeads Bromide content of the microbeads was found gravimetrically by silver bromide (AgBr) precipitation method [51]. In this procedure 0,4 g of the PBEMA microsphere was treated with 20 mL of methanolic NaOH solution (20 wt %) and refluxed for 24 h in the presence of a reflux condenser. Then the reaction mixture was filtered off with blue band filter paper and washed with distilled water (20 mL). All of the filtrate were collected in a 50 5
mL volumetric flask and the needed amount was completed with distilled water. Half of this filtrate was neutralized with nitric acid and mixed with excess amount of AgNO3 solution (1 M). The resulting AgBr precipitate was filtered off, washed with water and dried at 40 oC under atmospheric pressure. The AgBr was weighted as 0.057 ± 0.002 g and this indicated 0.76 mmol.g-1 of accessible bromide content.
2.5. Azidation of the PBEMA microspheres The bromide group on the PBEMA microbeads was substituted with azide ones by reaction with NaN3. For this purpose, the PBEMA microsphere (1 g) was mixed with NaN3 (0.325 g, 5 mmol) in 25 mL dimethylformamide (DMF) with a small amount of distilled water to enhance the solubility of sodium azide. The reaction mixture was sealed and covered with aluminum foil to avoid light exposure and shaken for 24 h at room temperature. The resulting PBEMA bead product was isolated by filtration and successive washings with water (30 mL × 2), methanol (30 mL) and dried overnight under vacuum at room temperature.
2.6. Synthesis of 1-[(2-Propynyloxy)methyl]pyrene](POMP) The propargyl pyrene was synthesized according to previous report [49]. 1- Pyrene methanol (1.16 g, 5 mmol) was reacted with sodium hydride (60 wt % dispersion in oil) (0.131 g, 5.5 mmol) in the presence of 20 mL dry THF and the reaction mixture was stirred at 0 oC under nitrogen for 1 h. Then propargyl bromide (0.654 g, 5.5 mmol) in toluene was added dropwise to the solution and the reaction carried out for 24 h at room temperature. After then the mixture was refluxed for 4 h in the dark. The reaction solution was extracted with ethyl acetate and half of the solvent evaporated. A light yellow product was obtained after the solvent removal. The raw product was dissolved in toluene and passed through the basic alumina column to remove unreacted 1-pyrene methanol. After removing toluene by evaporation, the residue was dried under vacuum oven (Yield: 58 %). 1H NMR (500 MHz, CDCl3) δ:8.00-8.42 (m, 9H, Ar H), 5.35 (s, 2H, CH2), 4.34 (d, 2H, CH2).
2.7. Preparation of the pyrene-linked PBEMA microspheres via click reaction The crosslinked PBEMA microspheres (0.2 g, containing 0.15 mmol azide groups) were dispersed in 4 mL DMF in a flask. 1-[(2-Propynyloxy)methyl]pyrene] (77 mg, 0.3 mmol), CuBr (22.8 mg, 0.15 mmol) and PMDETA (33.3 μL, 0.15 mmol) were then added to the mixture under inert atmosphere. The reaction was carried out at 40 oC with continuous 6
stirring for 48 h. The resulting pyrene-linked PBEMA microspheres were separated from the mixture by centrifugation. For the purification purpose, the resulting crude solid microbeads at room temperature was re-dispersed in DMF and separated by centrifugation. This purification step was repeated three times with dilute acidic methanol solution and THF. After purification, the resulting microbeads were dried overnight under vacuum and weighted as 215 mg.
2.8. Fluorescence detection procedure The PBEMA microsphere stock solution was prepared by dispersing 5 mg of microsphere in 100 mL of pure water. The suspension was sonicated for 1 hour. 4.4x10-4, 5.5x10-4 and 6x10-4 M of the stock solution of nitroaromatic compounds was prepared by adding 10, 10.1, 13.8 mg of TNT, DNT and TNP in 100 mL pure water. In each case, 3 mL of the PBEMA microspheres suspension (0,05mg/mL) was put into a quartz cuvette (1 cm length) with a magnetic stirrer and individually TNT, DNT and TNP aqueous solution (5μL) was added gradually onto the quartz cuvettes by using microliter syringe. The total concentration was changed from 0 to 11 μM. For each addition, at least three fluorescence spectrums were recorded at 25 oC to obtain a concordant value. The λ excitation was chosen 342 nm with 5 nm slit width. Note that the pyrene emissions (excimer at 480 nm, monomer at 396 nm) were quenched upon addition of nitroaromatics.
3.Results and discussion 3.1. Preparation of the pyrene functional crosslinked microbeads This work is aimed for the design and synthesis of dense pyrene-linked microbeads exhibiting an intense fluorescence for sensing and detection of nitro compounds in trace quantities. The synthesis pathway involves, i) Crosslinking copolymerization of 2-bromoethyl methacrylate with ethylene glycol dimethacrylate by the precipitation polymerization to give 2-5 µm size particles. The precipitation polymerization was deliberately chosen to attain high surface area-to-volume ratios. ii) in the second step the bromide substituents were replaced with azide groups. iii) Then, pyrene fluorophore was anchored to the micro-particle surfaces by alkyne-azide click chemistry using 1-[(2-Propynyloxy)methyl]pyrene] as depicted in Scheme 1. The bromide content of the microspheres was estimated to be 0.76 mmol/g by AgBr method. However according to feed composition in the polymerization, the real 7
bromide concentration should be around 5.0 mmol per gram. This implies that only 15.2 % of total bromide is accessible. Remaining portion, 85 % has been embedded in the crosslinked polymer matrix. The accessible portion must be located at the surfaces of the microspheres. This means that, accessible azide groups are located at the surfaces, which are amenable to click with POMP in the last step. The SEM picture also has showed no mechanical crack which implies retaining of the spherical shapes in the surface modifications (Supporting Information, Figure S1).
3.2 Examination of fluorescence behavior of the pyrene-linked microspheres The fluorescence (PL) properties of pyrene-linked microspheres were investigated using water as the dispersing medium. For comparison of the fluorescence behaviors, first, emission spectrum of 1-[(2-Propynyloxy)methyl]pyrene] was taken in aqueous dispersion.
this compound lie below 350 nm. These peaks are associated with Stocks shift of transitions appeared in its absorption spectrum. Whereas in PL emission spectrum of pyrene functional microspheres (Fig. 3 red) represents two broad bands centered at 396 nm and 480 nm. The strong emission at 480 nm that is assigned as excimer emission. This must be due to close proximity of surface bound pyrene moieties. Such a proximity facilitates ππ interaction of excited pyrene with another pyrene in ground state (excimer formation). In other words, the excimer emission band at 480 nm is observable even by naked eye and originates from luminescence of pyrene-excited pyrene conjugates. Therefore, organic compounds that are able to destroy this conjugate can quench excimer emission. Additionally, we have also investigated fluorescence lifetime of the pyrene tethered microbeads. For this purpose, 0.05mg/mL stock solution of the particles were used in this experiment. Results indicate, decaying one with 11.84 ±1.7 nanoseconds (ns) of lifetime and another one with 49.87 ± 1.17 ns of lifetime. Apparently, the longer lifetime represents the pyrene attached to inner part of the particle and the fast one shows emission of the pyrene located at the surfaces. Presumably this is due to easy energy transfer from excited outmost pyrenes to the solvent molecules. Stability of the pyrene linkage on the microbeads was also checked by simply monitoring of the excimer emission intensity in the presence and absence of the light. During the daylight interaction, fluorescence plot of the microbeads show decrease on the excimer emission band at around 480 nm due to the photo-decomposition of the ether linkages via light induced peroxide formation. In the same plot, an increment of free pyrene emission band were observed day by day due to the cleavage, as expected. Whereas, the samples stored in the dark did not show any noticeable decrease of PL emission. The results were collected in Fig. S9. In this work, we have investigated quenching effect of some aromatic nitro compounds on the green-blue excimer emission of surface-bound pyrene. This was achieved simply by adding 33 µL from stock solutions (1mM) of the TNT, DNT and TNP individually onto 3 mL aqueous dispersions of pyrene functional microspheres (0.05 mg/mL) while fluorescing. An immediate fading of the excimer emission was observed in all cases as depicted in Fig. S3 (see supporting information). This result clearly indicates high sensitivity of the pyrene-linked microspheres towards nitroaromatics. Quenching efficiencies (QE) of the nitro compounds in this condition were determined 86.7 %, 92.2 %, 85 % for TNT, TNP and DNT respectively. The quenching efficiencies (QE) were estimated based on the following relationship, 9
Io
100
where I0 and I are microsphere excimer fluorescence intensities at 480 nm in the absence and presence of nitroaromatics. QE of 2,4,6-trinitro phenol is somewhat higher than those of the others. Considering π electron densities, TNP having the lowest electron deficiency is expected to induce low or moderate extinction effect on the excimer emission. This controversy can be explained in terms of acid-base interaction between acidic OH group of 2,4,6-trinitro phenol and basic triazole connecting group. Three nitro groups on the phenol moiety make it much more acidic, so that TNP tends to form salt with triazole ring and this results in quick distraction of the excimer with 2,4,6-trinitro phenol.
3.3. Quenching of the excimer emission as a function of nitroaromatic concentrations Logically quenching efficiency of nitroaromatics must be concentration depended. To investigate effects of the quencher concentrations a series of experiments were performed, in which the stock solutions of TNT, TNP and DNT were gradually added into pyrene-linked microsphere dispersions (0.05 mg/mL) using microliter syringes.
’s,
however there is no linear relationship. The highest QE is attained by TNP and reaches to 92.2 for the concentration of 50 µM as discussed above. Further fluorescence behavior of pyrene functional microspheres was explored in the presence of various nitro compound 4nitrotoluene (4-NT), nitromethane (NM), nitrophenol (NP) and nitrobenzene (NB). It can be seen that there was no significant effect on fluorescence intensity. The results suggest that the fluorescent microspheres are highly selective for explosive detection.
where I0 and I are fluorescence intensities in the absence and presence of a quencher and [Q] represents the quencher concentration. Ksv denotes Stern-Volmer constant which depends on solvent and nature of the quencher and fluorescence source. Linearity of I0 /I versus [Q] plots implies validity of the Stern-Volmer quenching mechanism for the nitroaromatics studied. The regression factors (R2) were obtained as 0.991, 0.981, and 0.988 for TNT, TNP, and DNT respectively. Closeness of the regression factors to unity indicates good linearity in all cases. Stern-Volmer constants obtained from the slopes of curves were determined the 1.33×105, 2.451×105 and 1.076×105 M-1 for TNT, TNP, and DNT respectively. Those high Ksv values imply possibility of precise estimation of nitroaromatics by this system. The highest Ksv for the case of 2,4,6-trinitro phenol indicates its extreme sensitivity in quenching of the excimer emission. The detection limits of TNT, TNP and DNT have been determined to be 2.1×10-7 M, 1.14×10-7 M and 2.6×10-7 M respectively, one of the most sensitive sensors that have been reported and used in aqueous solution (Supporting information, Table S1). We also demonstrated a simple solid phase detection of TNT and TNP via adding different concentration on microspheres adsorbed TLC plate (Shown in Fig.S5 and Fig.S6 in Supplementary Material).
3.4. The solvent effect Solvent effect is known as an important parameter for both the fluorescence intensity and quenching. Excimer formation must also be dependent on solvent type. According to the literature there is a strong relationship between the excimer formation and the solvent polarity [53]. To investigate the solvent effect in this work, the fluorescence spectra of pyrene-linked microspheres were recorded while contacting of the microspheres with different solvents. All the spectra were collected in Fig. 6.
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In the case of THF and acetonitrile, intensity of the emission band at 396 nm (monomer emission) is greater than that of the excimer emission. This could be explained by good solvent behavior of THF and acetonitrile on both the pyrene moieties and the naked PBEMA microbeads. After the solvents interaction with the microbeads, swollen microbeads may cause the distance increment between the anchored pyrene groups on the surface of the beads and decreasing of the excimer formation. In the methanol and water cases, the intensity of the excimer emission band is significantly higher than the monomer emission band. These two solvents are not good solvent for both pyrene groups tethered on the surface of the beads and the naked PBEMA beads. So the distance decreases between the pyrene moieties due to the shrinkage of the PBEMA beads and this cause to increase excimer formation.
3.5. Reversibility of the quenching process The effective sensing properties of microsphere were also supported by regeneration of the immobilized pyrenes. TNT was the only quencher used in the regeneration experiments. For this purpose, a UV cell was filled with 3 mL of the microsphere dispersion and aqueous TNT solution (10 μM) was added to the dispersion and the excimer emission was measured. The microspheres were separated by centrifugation and dried at atmospheric pressure at 50 oC for overnight. Being very soluble in acetone, TNT residue was removed by washing the microspheres with 10 mL of acetone twice and dried. Then the excimer intensity was measured once again. The microspheres were recovered by washing with acetone and reused as excimer emission source in the second time. These cycles were repeated for many times and the intensities of the excimer emission were noted. The excimer band intensities were plotted against cycling numbers as seen in Fig. S8. The figure reveals almost constant excimer emission intensity throughout the regeneration cycles. Slight decrease of the excimer emission intensity might be due to mechanical loss of the particles. In another word, it is clear that this sensing process is reversible and recovered particles show good retention of the sensitivity towards aromatic nitro compounds. Reversibility was also observed in solid phase visually (Supplementary Material, Fig.S4).
4. Conclusions Pyrene tethered crosslinked microspheres were prepared by anchoring of 1-[(2Propynyloxy)methyl]pyrene] onto the surface of the microspheres via alkyne-azide click 12
chemistry. Surface bound pyrene moieties tend to form excimer in methanol, water and acetonitrile. This fact stimulates fluorescing a strong and green-blue excimer emission with a maximum at 480 nm. The excimer emission was determined to be very sensitive to TNT, TNP and DNT with very low limit of detection 2,1×10-7 M, 1,14×10-7 M and 2,6×10-7 M respectively. The nitroaromatic contaminants on microspheres can be removed by simple washing with acetone and the resulting particles are reusable many times for precise detection of these nitro compounds. Having these peculiarities, the presented system provides excellent platform for easy detection of explosives even in trace quantities.
Acknowledgements The authors are thankful to Istanbul Technical University graduate school of science engineering and technology for the financial support. We are also grateful to Prof. Dr. Ismail Yilmaz for his helps in taking fluorescence spectra. We are also thankful to YTU Department of Chemistry for fluorescence lifetime measurement.
Appendix A. Supporting Information Various type of microscope images, UV-Vis absorbance spectra and solid state visual detection studies are associated with this article given on supplementary material.
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Figure Captions Fig. 1. FTIR spectra of a) 2-Bromoethyl methacrylate polymer (PBEMA) b) the azide functionalized PBEMA c) the pyrene functionalized PBEMA. Fig. 2. Fluorescence microscope image of the pyrene-linked microspheres showing uniformly distributed size and fluorescence intensities. Fig. 3. Fluorescence emission spectra of free (Black) 1-[(2-Propynyloxy)methyl]pyrene] (2x10-6 M) and the pyrene-linked PBEMA microspheres (Red) (0,05 mg/mL) in water. Fig. 4. Quenching percentage of pyrene functional microspheres for various nitro compounds. (The concentrations varied 0-50 μM) range. Pyrene-linked micro-particles 0.05 mg/mL. λex =342 nm, ex/em slit widths = 5/5 nm). Fig. 5. Full PL spectra of the pyrene-linked microspheres upon the addition of TNT, TNP, and DNT in 100 % aqueous solution (λex 342 nm). The inset is its corresponding SternVolmer plot. Fig. 6. Fluorescence emission spectra of the pyrene anchored microspheres in the presence of different solvents.
Scheme Scheme 1. Preparation of highly fluorescent the pyrene-linked polymer starting from densely crosslinked 2-bromoethyl methacrylate polymer, for detection of nitroaromatics in trace quantities and their SEM images.
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