Fluorescence Turn-On Sensing of Anions Based on Disassembly

Article pubs.acs.org/Macromolecules

Fluorescence Turn-On Sensing of Anions Based on Disassembly Process of Urea-Functionalized Poly(phenylenebutadiynylene) Aggregates Ryosuke Sakai,†,§ Atsushi Nagai,† Yasuyuki Tago,† Shin-ichiro Sato,† Yoshinobu Nishimura,‡ Tatsuo Arai,‡ Toshifumi Satoh,† and Toyoji Kakuchi*,† †

Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, Sapporo 060-8628, Japan ‡ Graduate School of Pure and Applied Chemistry, University of Tsukuba, Tsukuba 305-8571, Japan S Supporting Information *

ABSTRACT: A rationally designed poly(phenylenebutadiynylene) bearing urea functionalities (poly1) was demonstrated to be a novel fluorescence turn-on probe for various anions. Poly-1 itself exhibits an extremely weak emission that was undetectable by the naked eye, whereas the fluorescence emission of poly-1 was significantly enhanced by the addition of anions. Based on the NMR, dynamic light scattering, and fluorescence decay measurements, the observed fluorescence turn-on response was determined to be realized by the fluorescence recovery based on the disassembly of the poly-1 aggregates, which was originally triggered by the anion recognition event at the urea units of poly-1.

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INTRODUCTION A reliable, robust, and efficient anion sensor has been increasingly demanded because anions are significant analytes in diverse fields including the environmental, industrial, biological, and medical ones.1 Thus, a number of fluorescent and colorimetric probes for anions have been developed toward practical use, in which fluorescence and colorimetric changes, fluorescence enhancement, and fluorescence quenching techniques have been employed as detection strategies.2−12 In chemical sensor developments, fluorescence turn-on sensing has received a great deal of attention because fluorescent OFF/ ON switching is much easier to discriminate than other detection approaches.13−17 Therefore, considerable effort has been devoted recently to fabricating fluorescence turn-on probes for anion sensing.18−22 For example, Schanze and colleague have succeeded in selective fluorescence turn-on sensing of pyrophosphate by using the complex consisting of carboxylate groups-conjugated poly(phenyleneethynylene) and Cu2+ cation, in which the original florescence of the conjugated polymer is recovered by the competing Cu2+/pyrophosphate complex formation.18 Hamachi and co-workers have reported that a xanthene-based Zn(II) complex exhibits fluorescence turn-on response to a series of nucleoside polyphosphates through the binding-induced recovery of the conjugated form of the xanthene ring.19 Lee et al. have realized a reaction-based cyanide sensor with turn-on fluorescence response, in which nucleophilic attack of cyanide ion on the carbonyl group of latent fluorophore molecule produces a fluorescence signaling property.21 Although such florescence turn-on probes are useful © 2012 American Chemical Society

even for practical anion sensing, the applicable anions are limited to certain anions with a strong basicity and/or nucleophilicity because of the employed detection mechanism such as reaction-based sensing and indicator displacement assay. Therefore, the realization of a fluorescence turn-on probe for a wide range of anions is still desired.23 We now report the development of a novel fluorescence turn-on probe for anions, in which the sensing mechanism is based on a fluorescence recovery driven by the disassembly process of self-assembling fluorescent polymers through anion recognition. For this purpose, we have designed a ureafunctionalized poly(phenylenebutadiynylene) (poly-1), which emerges as an ideal candidate due to the following expectations (Figure 1). Poly-1 would self-assemble to form aggregates through intermolecular hydrogen bonding between the urea units,24−27 which might collapse in response to an anion binding event at the urea units of poly-1. This disassembly process is anticipated to trigger the recovery of the original fluorescence, thus realizing a fluorescence turn-on response.28−31 To prove the feasibility of such a concept, fluorescence, NMR, dynamic light scattering (DLS), and fluorescence decay measurements of poly-1 were carried out in the presence of anions. Received: March 8, 2012 Revised: May 1, 2012 Published: May 8, 2012 4122

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Figure 1. Schematic illustration of fluorescence turn-on sensing of anions based on the disassembly of poly-1 aggregates.

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RESULTS AND DISCUSSION The copper(I)-mediated oxidative coupling polymerization of 1-[3,5-bis(trifluoromethyl)phenyl]-3-(2,4-diethynylphenyl)urea (1) was carried out at 60 °C with air bubbling for 24 h to produce urea-functionalized poly(phenylenebutadiynylene) (poly-1) in 64% yield (see Supporting Information).32,33 The size exclusion chromatography (SEC) measurement estimated the number-average molecular weight (Mn,SEC) and molecular weight distribution (Mw/Mn) of poly-1, which were 1.4 × 104 and 2.6, respectively. The anion-sensing ability of poly-1 was evaluated based on its fluorescence property in the presence of the tetra-nbutylammonium (TBA) salts of a series of anions in THF containing 0.1 vol % DMSO. Figure 2a shows the fluorescence spectra of poly-1 in the absence and presence of F−, which were obtained 24 h after the sample preparation because of the aftermentioned reason. The fluorescence spectrum of poly-1 itself exhibits an extremely weak emission with a maximum at 460 nm, which was undetectable by the naked eye. In sharp contrast, a drastic enhancement of the fluorescence emission was observed with the F− addition, which was greater than a 10fold increase. Moreover, the F− addition brought about a clear red-shift of the fluorescence emission band, which suggested different fluorescence behaviors. These results demonstrated that poly-1 possesses a fluorescence turn-on response ability to F−. The important point is that the above fluorescence measurements were carried out 24 h after the sample solution preparation. This is due to the fact that a temporal change in the fluorescence intensity was observed. The time dependence of the fluorescence spectra of poly-1 in the absence and presence of F− is shown in Figure S1 of the Supporting Information. The fluorescence intensity of the poly-1 with F− increased with time and reached an almost constant value in 20 h of the preparation of the poly-1/F− mixed solution (Figure 2b). On the other hand, the solution of poly-1 itself also showed a similar temporal change in the fluorescence intensity

after poly-1 was dissolved in the solvent. However, the timedependent fluorescence increase of poly-1 itself was much lower than that of the poly-1/F− mixed system, indicating that the F− addition is essential for the strong fluorescence enhancement. It is obvious that the F− addition would exponentially accelerate certain kinds of temporal structure changes in poly-1, resulting in the fluorescence turn-on response. To confirm the role and involvement of F− in this fluorescence turn-on response, we performed an 1H NMR titration experiment on monomer 1 with F−, in which 1 was used as a model compound of poly-1 because the highly broadened signals in the 1H NMR spectrum of poly-1 were expected to provide no useful information. With the addition of F−, a remarkable broadening and downfield shift was observed in the signals due to the urea protons of 1 (Figure 3). The downfield shift of the urea protons has been revealed to reflect the establishment of a hydrogen bond interaction between the urea group and anion.34 Therefore, F− was confirmed to bind to the urea groups on poly-1 through the hydrogen bond interaction, which should trigger structural changes in poly-1. To explain this fluorescence turn-on response, dynamic light scattering (DLS) measurements of poly-1 were performed in the presence and absence of F− (Figure 4). Poly-1 was confirmed to form aggregates in solution, of which the diameter was determined to be 25.3 nm. Given the actual performance of the urea functionality in the supramolecular chemistry field, the aggregates would be constructed by the intermolecularly formed hydrogen bonding between urea units.24−27 Similar to the fluorescence emission, a temporal variation in the particle size of the aggregate was observed; i.e., the particle size decreased from 25.3 to 3.6 nm 24 h after the solution preparation. This indicates the slow disassembly of the aggregates with time. On the other hand, no aggregates were detected in the DLS measurement of the poly-1 solution after the addition of F−. Therefore, the F− addition was determined to trigger the complete collapse of the self-assembly of poly-1, 4123

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Figure 3. Changes in the 1H NMR spectra of 1 upon the addition of 0−0.5 equiv of F− in DMSO-d6. The closed circles show the signals due to the urea protons.

response function (IRF) and fitted to the following equation consisting of three exponential terms by the iterative nonlinear least-squares method. I(t ) = A1 exp( −t /τ1) + A 2 exp(−t /τ2) + A3 exp(−t /τ3)

The fitting parameters are summarized in Table 1. Given the above fluorescence and DLS measurements, the shortest lifetime τ1 will reflect the fluorescence quenching due to the entanglement of the polymer chains for the aggregates of poly1. On the other hand, the longest lifetime τ3 would be identified with the original lifetime of the isolated polymer chain. Furthermore, the medium lifetime τ2 might be the loose clusters of poly-1. The amplitude of each lifetime component (An) represents the fractional contribution to the total fluorescence decay. In the absence of F−, the total fluorescence decay of poly-1 mainly consists of the shortest lifetime component τ 1 corresponding to the fluorescence quenching. This is the reason why the poly-1 emits almost no fluorescence before the F− addition. An obvious decrease in the contribution of the τ1 component (A1) was observed with the addition of F− at all the monitoring wavelengths, indicating that the F−-triggered disassembly of the aggregates produced the dissolution of the self-quenching. Along with this change, the contribution of the τ2 and τ3 components increased instead. In particular, the F− addition brought about a significant increase in the A3 values at 500 and 550 nm, which led to the recovery of the original fluorescence emission of the isolated polymer chain of poly-1. Therefore, the anion-triggered fluorescence turn-on event of poly-1 would be the direct consequence of the significant fluorescence recovery through the disassembly process of the aggregates.35 On the basis of all these results, we can comprehensively consider the anion-induced fluorescence turn-on process of poly-1 as follows: (1) Poly-1 forms aggregates in solution by intermolecular hydrogen bonding between the urea units, (2) the aggregates exhibit a negligible fluorescence emission due to the fluorescence quenching, (3) the F− addition produces disassembly of the aggregates through the complex formation between the urea unit and anion, and (4) the resulting isolated

Figure 2. (a) Fluorescence spectra of poly-1 in the presence and absence of F−. The measurements were conducted 24 h after the sample preparation. (b) Time dependence of the fluorescence intensity of poly-1 and poly-1/F− system at 493 nm. The measurements were carried out in THF containing 0.1 vol % DMSO at 25 °C (λex = 350 nm). The concentrations of the monomer units of poly-1 and anions were 13 μM and 1.3 mM, respectively.

in which the binding mode at the urea functionality probably changes from the intermolecular hydrogen-bonding interaction between the urea units to the urea/F− complex formation. Considering that the fluorescence quenching has often been observed in aggregates, the fluorescence and DLS results suggest that the observed fluorescence turn-on phenomenon is based on the recovery of the original fluorescence of poly-1 through the disassembly process. In order to provide direct evidence for this hypothesis in the fluorescence turn-on mechanism, the fluorescence decay measurements were performed using a time-correlated singlephoton counting technique, in which the fluorescence decay was monitored at 450, 500, and 550 nm. Figure 5 displays the resulting fluorescence decay curves of poly-1 before and after the F− addition. The decay curves cannot be fitted by a single exponential because it contains several decay components ranging from picosecond to nanosecond regions. Thus, the fluorescence decays were deconvoluted with the instrumental 4124

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Figure 4. Particle size distribution and average hydrodynamic diameter estimated of dynamic light scattering (DLS) measurements of poly-1 in THF containing 0.1 vol % of DMSO at 25 °C ([monomer units of poly-1] was 13 μM). The measurements were conducted (a) 1 and (b) 24 h after the polymer solution preparation. The diameter and size distribution were calculated using the histogram methods including the Marquadt analysis. Note: negligible intensity was obtained in the DLS measurement of poly-1 containing 1.3 mM of F−, which was performed 24 h after the solution preparation under the above condition.

polymer chain of poly-1 recovers the original fluorescence emission (Figure 1). To elucidate the applicability of this turn-on fluorescence sensing, we performed the fluorescence measurements of poly1 in the presence of various anions. As described above, the fluorescence emission of poly-1 itself was hardly observed by the naked eye. In sharp contrast, the polymer solutions exhibited bright fluorescent emissions by the addition of anions except for ClO4− (Figure 6a). Thus, poly-1 was found to be utilizable as a fluorescent turn-on probe for a wide range of anions. This wide applicability would be achieved by making use of an anion−urea complex formation as a detection mechanism instead of previously reported reaction-based sensing and indicator displacement assay. Interestingly, the observed anion-induced fluorescence changes were dependent on the type of anions (Figure 6b). Given that the binding

Figure 5. Fluorescence decay curves of poly-1 in a THF/DMSO (99/ 1 (v/v)) mixed solution at room temperature. The fluorescence decay was monitored at (a) 450, (b) 500, and (c) 550 nm. The concentrations of the monomer units of poly-1 and F− were 13 μM and 1.3 mM, respectively. The measurements were conducted immediately for poly-1 and 24 h after the sample preparation for the poly-1/F− system.

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Table 1. Fitting Parameters for Fluorescence Decay Curvesa lifetime and its amplitude sample poly-1

poly-1/F−

λobs (nm)

τ1 (ps)

A1

τ2 (ns)

A2

τ3 (ns)

A3

χ2 b

450 500 550 450 500 550

44.1 79.4 105 174 250 245

0.814 0.802 0.793 0.574 0.434 0.443

1.05 1.57 1.49 0.895 1.55 1.77

0.103 0.150 0.163 0.342 0.261 0.285

3.81 7.68 8.67 3.26 4.57 4.97

0.083 0.048 0.044 0.084 0.305 0.272

1.03 1.19 1.09 1.08 1.02 1.01

a

Fluorescence decay measurements were performed in a THF/DMSO (99/1 (v/v)) mixed solution at room temperature. The concentrations of monomer units of poly-1 and F− were 13 μM and 1.3 mM, respectively. The measurements were conducted immediately for poly-1 and 24 h after the sample preparation for the poly-1/F− system. bChi-squared test of the fitting curve.

detection sensitivity is probably due to the adopted fluorescent method instead of the colorimetric one.

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CONCLUSIONS

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ASSOCIATED CONTENT

We have developed a novel fluorescence turn-on probe for anion sensing, which utilizes the urea-functionalized poly(phenylenebutadiynylene) (poly-1). Based on various experiments, the observed fluorescence turn-on property was clarified to be realized by the anion-recognition triggered disassembly process of the poly-1 aggregates. Given the anion receptors that have already been fabricated, the demonstrated disassembly based fluorescence turn-on detection is a novel strategy and approach for anion sensing, thus expanding the limit and scope of anion recognition chemistry. In addition, the fluorescence turn-on sensing definitely has significant advantages in discriminability by the naked-eye compared to sensing based on a fluorescence enhancement and fluorescence shift. Although the time required for the fluorescent detection is a weak point for practical use, we believe that the modulability in the polymer design allows producing an ideal fluorescence turnon probe for anion detection with a sufficient response speed. Therefore, the demonstrated fluorescence turn-on detection of anions will contribute to the future design of reliable and sophisticated anion sensor materials.

Figure 6. (a) Photograph and (b) fluorescence spectra of poly-1 in the presence of TBA salts for a series of anions in THF containing 0.1 vol % of DMSO at 25 °C. The excitation wavelengths were 365 nm for the photograph and 350 nm for the spectra. The concentrations of the monomer units of poly-1 and anions were 13 μM and 1.3 mM, respectively. The measurements were conducted 24 h after the sample preparation.

S Supporting Information *

Experimental details and Figures S1−S3. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

ability of the urea receptor is generally dictated by the basicity of the anions,2,36 the fluorescence changes of poly-1 were considered to be dependent on the basicity of the anions.37 Although poly-1 seems to detect almost all the above anions without a strict selectivity under this condition, the multiplicity of the fluorescent emission allows a rough discrimination of the anions by the naked eye. Finally, we carried out a fluorescence titration experiment for the poly-1/F− system to determine the detection sensitivity (Figure S3 in Supporting Information). The fluorescence intensity at 493 nm, which was self-quenched in the absence of F−, significantly increased with the increasing added amount of F−. The spectral change suggested that the detection limit of poly-1 for F− was less than 5 μM. This detection sensitivity is greater than 10 times that of colorimetric anion sensors that we have previously fabricated.38−41 The improvement of the

*Tel +81 11 706 6602; Fax +81 11 706 6602; e-mail kakuchi@ poly-bm.eng.hokudai.ac.jp. Present Address §

Department of Materials Chemistry, Asahikawa National College of Technology, Asahikawa 071-8142, Japan.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid for Scientific Research (B), a Grant-in-Aid for Young Scientists (B), and the Global COE Program (Catalysis as the Basis for Innovation in Materials Science) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan. 4126

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that the observed fluorescence emissions around 450 and 500 nm correspond to the loose clusters and the isolated chain of poly-1, respectively. (36) Amendola, V.; Esteban-Gomez, D.; Fabbrizzi, L.; Licchelli, M. Acc. Chem. Res. 2006, 39, 343−353. (37) UV−vis absorption measurements of poly-1 were additionally performed in the presence of a series of anions (Figure S2 in Supporting Information). Bathochromic shifts were observed for F−, AcO−, BzO−, and N3−, which are anions that produced bright green emissions. In contrast, Cl−, Br−, and NO3−, which induced blue fluorescence emissions, exhibited no essential absorption changes. Thus, the difference between the green emission and blue one seemed to be related to the absorption changes. (38) Kakuchi, R.; Nagata, S.; Sakai, R.; Otsuka, I.; Nakade, H.; Satoh, T.; Kakuchi, T. Chem.Eur. J. 2008, 14, 10259−10266. (39) Kakuchi, R.; Tago, Y.; Sakai, R.; Satoh, T.; Kakuchi, T. Macromolecules 2009, 42, 4430−4435. (40) Sakai, R.; Okade, S.; Barasa, E. B.; Kakuchi, R.; Ziabka, M.; Umeda, S.; Tsuda, K.; Satoh, T.; Kakuchi, T. Macromolecules 2010, 43, 7406−7411. (41) Sakai, R.; Sakai, N.; Satoh, T.; Li, W.; Zhang, A.; Kakuchi, T. Macromolecules 2011, 44, 4249−4257.

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