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A Hemicyanine-Conjugated Copolymer as a Highly Sensitive Fluorescent Thermometer Yasuhiro Shiraishi,* Ryo Miyamoto, and Takayuki Hirai Research Center for Solar Energy Chemistry, and DiVision of Chemical Engineering, Graduate School of Engineering Science, Osaka UniVersity, Toyonaka 560-8531, Japan ReceiVed December 13, 2007. In Final Form: January 12, 2008 A simple-structured copolymer, poly(NIPAM-co-HC), consisting of N-isopropylacrylamide (NIPAM) and 4-(4dimethylaminostyryl)pyridine (hemicyanine, HC) units as thermoresponsive and fluorescent signaling parts, respectively, has been synthesized. This copolymer dissolved in water shows very weak fluorescence at 25 °C. The fluorescence intensity increases with a rise in temperature and saturates at >40 °C, enabling temperature detection at 25-40 °C. The fluorescence enhancement is driven by a heat-induced phase transition of the polymer from coil to globule state. The HC units within the coil state polymer exist as the nonfluorescent benzenoid form; however, the less polar domain formed inside the globule state polymer leads to transformation of the HC unit to the fluorescent quinoid form, resulting in heat-induced fluorescence enhancement. The fluorescence intensity measured at 40 °C is >20-fold higher than the intensity at 40 °C. The fluorescence intensity at 40 °C is 35fold higher than the intensity at 25 °C is driven by a heat-induced phase transition of the polymer from coil to globule state containing a less polar domain. Figure 4 (black) shows a change in turbidity (A700nm) of the polymer solution determined from the absorption spectra (Figure 1B). The turbidity increases drastically at >31 °C, indicating that strong polymer aggregation (formation of multi chain globule) takes place at >31 °C. The temperature-turbidity profile is similar to the temperature-fluorescence intensity profile (Figure 3a), indicating that the formation of the less polar domain within the globule state polymer triggers the fluorescence enhancement of the HC unit. The formation of the less polar domain is confirmed by 1H NMR analysis. Figure 5A shows a change in 1H NMR spectra of poly(NIPAM-co-HC) dissolved in D2O (pH 6.6). Temperature-dependent change in the integrated proton intensity
Figure 5. (A) Temperature-dependent change in partial 1H NMR spectra of poly(NIPAM-co-HC) dissolved in D2O (pH 6.6). (B) Change in the integrated proton intensity of CH resonances of the polymer chain and the NIPAM units as a function of temperature. The integrated CH proton intensity at 15 °C is set as 1.
of the CH resonances of the polymer chain and the NIPAM units is shown in Figure 5B. The proton intensity decreases drastically at >31 °C, suggesting that the less polar domain actually forms.9 The proton intensity decrease is consistent with the drastic fluorescence enhancement (Figure 3a) and the turbidity increase (Figure 4, black). These findings strongly suggest that the formation of the less polar domain associated with the polymer aggregation triggers the fluorescence enhancement of the HC unit. As shown in Figure 3a, the fluorescence intensity slightly increases at 25-31 °C. This is because weak aggregation of the polymer leads to a polarity decrease around the HC unit;10a,19 as shown in Figure 5B, the proton intensity gradually decreases at 15-31 °C. As shown by photographs in Figure 2b, the polymer
A Hemicyanine-Based Fluorescent Thermometer
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Table 1. Solvent Parameters and Photophysical Properties of 1 at 25 °Ca
entry
solvent
b
nb
∆f
absorption
emission
Stokes shift (cm-1)
a b c d e f g h i
water methanol CH3CN ethanol acetone DMF DMSO CH2Cl2 CHCl3
80.4 33.6 37.5 34.9 20.7 38.3 46.4 8.9 4.8
1.333 1.328 1.344 1.361 1.359 1.431 1.482 1.424 1.445
0.319 0.308 0.305 0.289 0.284 0.274 0.263 0.217 0.149
449 475 471 481 472 470 471 518 499
584 581 596 582 595 596 598 593 564
7407 9434 8000 9901 8130 7937 8475 13333 15385
λmax (nm)
φF 0.0009 0.0014 0.0010 0.0056 0.0013 0.0015 0.0048 0.0152 0.0186
a The fluorescence and absorption spectra of 1 measured in respective solvents are summarized in Figure S5 of the Supporting Information.18 b The values are from ref 22.
summarizes the maximum wavelengths of the absorption and fluorescence spectra of the reference compound 1 measured in various solvents. Figure 6A summarizes the relationship between the Stokes shift and the solvent polarity parameter (∆f), determined with the following equation,21
∆f )
(
-1 n2 - 1 - 2 2 + 1 2n + 1
)
(1)
where is the dielectric constant and n is the refractive index of the solvents.22 The Stokes shift increases linearly with a decrease in solvent polarity. This clearly indicates that the quinoid form predominates with the polarity decrease. Figure 6B summarizes the relationship between the fluorescence quantum yield (φF) of compound 1 and the solvent polarity parameter, where the φF value is determined with the following equation,23
φF ) φFR Figure 6. The relationship between the solvent polarity parameter (∆f) and (A) Stokes shift and (B) fluorescence quantum yield (φF) of 1 measured in respective solvents (a-i). The solvents a-i correspond to those in Table 1. The fluorescence and absorption spectra of 1 are summarized in Figure S5 of the Supporting Information.18
solution is homogeneous at low temperature (left), but small particles appear at >30 °C (right) due to the polymer aggregation. Figure 4 (white) shows a change in hydrodynamic radius (Rh) of the polymer particles measured by laser scattering analysis. At 30 °C, polymer particles appear and the size of the particles increases gradually with a rise in temperature. At 40 °C, the size of the particles is estimated to be 11 µm. The fluorescence enhancement of the polymer is triggered by a change in the resonance structure of the HC unit from the benzenoid to the quinoid form, associated with the heat-induced decrease in the inner polarity of the polymer. As described,20 the HC molecule shows a red shift of the absorption spectrum and a blue shift of the fluorescence spectrum with a decrease in solvent polarity, which is associated with the change in resonance structures (Scheme 1). The Stokes shift (cm-1) between the absorbance and fluorescence maxima of the HC molecule can therefore be correlated with the solvent polarity.15 Table 1 (20) (a) Wang, H.; Helgeson, R.; Ma, B.; Wudl, F. J. Org. Chem. 2000, 65, 5862-5867. (b)Huang, Y.; Cheng, T.; Li, F.; Luo, C.; Huang, C.-H.; Cai, Z.; Zeng, X.; Zhou, J. J. Phys. Chem. B 2002, 106, 10031-10040. (c) Wang, S.-L.; Ho, T.-I. Chem. Phys. Lett. 1997, 268, 434-438. (d) Mishra, A.; Haram, N. S. 2004, 63, 191-202.
( )( ) FAR n FRA nR
2
(2)
where φFR is the fluorescence quantum yield of the reference compound (anthracene, 0.297 in ethanol). F and FR are the integrated values of the fluorescence spectra for the sample and reference, A and AR are the absorbance at the excitation wavelength (510 nm), and n and nR are the refractive indexes of the solvents.23 The φF value increases linearly with a decrease in the solvent polarity (Figure 6B). The above findings for the compound 1 clearly suggest that the decrease in solvent polarity triggers the structure change from the benzenoid to the quinoid form, resulting in fluorescence enhancement. The Stokes shift and φF value of poly(NIPAM-co-HC) measured in water at 20 °C were determined to be 8475 cm-1 and 0.001, respectively. Comparison of these values with the data for compound 1 (Figure 6) suggests that the polymer actually has a polar character at low temperature. As a result of this, the HC units exist as the benzenoid form, resulting in low fluorescence intensity at 25 °C suggests that the polymer aggregation leads to inner polarity decrease, resulting in transformation of the HC unit to the fluorescent quinoid form. As shown in Figure 7, the fluorescence intensity of the polymer (21) (a) Hayakawa, J.; Ikegami, M.; Mizutani, T, Wahadoszamen, Md.; Momotake, A.; Nishimura, Y.; Arai, T. J. Phys. Chem. A 2006, 110, 1256612571. (b) Chakraborty, A.; Kar, S.; Guchhait, N. J. Photochem. Photobiol. A 2006, 181, 246-256. (c) Zoon, P. D.; Brouwer, A. M. Chem. Phys. Chem. 2005, 6, 1574-1580. (d) Benniston, A. C.; Harriman, A.; Roston, J. P. Phys. Chem. Chem. Phys. 2005, 7, 3041-3047. (22) (a) Griffiths, T. R.; Pugh, D. C. Coord. Chem. ReV. 1979, 29, 129-211. (b) Chastrette, M.; Rajzmann, M.; Chanon, M.; Purcell, K. F. J. Am. Chem. Soc. 1985, 107, 1-11. (23) (a) Bag, B.; Bharadwaj, P. K. J. Phys. Chem. B 2005, 109, 4377-4390. (b) Proutiere, A.; Megnassan, E.; Hucteau, H. J. Phys. Chem. 1992, 96, 34853489.
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Figure 7. Fluorescence spectra (λexc ) 510 nm) of 1 (13.6 µM) measured in respective solvents (a-i) at 25 °C and of (bold lines) poly(NIPAM-co-HC) measured in water (0.1 g L-1 containing 13.6 µM HC unit) at 20 and 40 °C. The solvents a-i correspond to those in Table 1. The fluorescence intensity of poly(NIPAM-co-HC) measured at 15 °C is set as 1. Scheme 3. Heat-Induced Fluorescence Enhancement Mechanism of Poly(NIPAM-co-HC) in Water
Figure 8. (A) pH-dependent change in the structure of 1. pHdependent change in (B) absorption and (C) fluorescence spectra (λexc ) 510 nm) of 1 in water (13.7 µM) measured at 25 °C.
measured at 40 °C (bold line) is much higher than that of 1 measured in various organic solvents, such as ethanol (d), DMSO (g), and DMF (f). This indicates that the inner polarity of the globule state polymer is significantly low. The overall fluorescence enhancement mechanism of poly(NIPAM-co-HC) can be summarized as Scheme 3: the heat-induced polymer aggregation provides the less polar domain inside the polymer, which leads to transformation of the HC unit from the benzenoid to the fluorescent quinoid form, resulting in strong fluorescence enhancement. It must be noted that copolymerization of the HC unit with polyNIPAM is necessary for the onset of the heat-induced fluorescence enhancement. Figure 3c shows the temperaturedependent change in the fluorescence intensity of 1 (13.7 µM) measured with a HC-free polyNIPAM (0.1 g L-1) in water (pH 6.3). The fluorescence intensity increases at >31 °C, but the intensity is much lower than that of poly(NIPAM-co-HC) (Figure 3a). The temperature-dependent change in turbidity and hydrodynamic radius of the HC-free polyNIPAM (Figure S618) is similar to that of poly(NIPAM-co-HC) (Figure 4), indicating that the aggregation behaviors of these polymers are similar. The very low fluorescence enhancement of 1 with HC-free polyNIPAM is probably because the bulky HC molecule is not
encapsulated sufficiently within the less polar domain of the globule state of the polymer.10a,19 A notable feature of this polymeric thermometer is that it can be used at wide pH range (2-12). It is well-known that, as shown in Figure 8A, the HC molecule is transformed to a nonfluorescent dication form by protonation of the aniline nitrogen at low pH.24 Figure 8B shows the pH-dependent change in absorption spectra of compound 1. A 350-550 nm absorption, assigned to the monocation form of 1, decreases with a pH decrease, along with an increase in 280-350 nm absorption, indicating that the dication form predominates at acidic pH (pKa 3.8).14 As shown in Figure 8C, the fluorescence intensity of 1 decreases with a pH decrease; the intensity becomes almost zero at pH 2. Poly(NIPAM-co-HC) measured at 20 °C also shows a dication formation at acidic pH (pKa 3.5) (Figure S718). Figure 9A shows the pH-dependent change in the fluorescence intensity of poly(NIPAM-co-HC) at 40 °C. Astoundingly, the fluorescence intensity is still high, even at acidic pH, although the intensity decreases with a pH decrease. As shown in Figures 9B and 1C, the shape of the excitation spectra collected at 580 nm is similar over the entire pH range, indicating that the quinoid form still exists even at acidic pH. As described,24 in less polar media, the dication form of the HC molecule is transformed to the monocation form by deprotonation of the aniline nitrogen. The high (24) Chi, L. F.; Dhathathreyan, A.; Mo¨bius, D. Langmuir 1990, 6, 13601363.
A Hemicyanine-Based Fluorescent Thermometer
Figure 9. (A) pH-dependent change in fluorescence spectra (λexc) 510 nm) of poly(NIPAM-co-HC) (0.1 g L-1) measured in water at 40 °C. (B) Change in excitation spectra (λem ) 580 nm).
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nitrogen of the HC unit by the less polar domain within the globule state polymer. As shown in Figure 3b, the temperaturefluorescence intensity profile of the polymer measured at pH 2.0 is similar to that obtained at neutral pH (Figure 3a). Although the intensity enhancement is relatively low (22-fold) at pH 2.0, the value is still higher than that of the early reported fluorescent thermometers.6,7 These findings suggest that the polymer behaves as a highly sensitive fluorescent thermometer over a wide pH range. The fluorescence enhancement/quenching of poly(NIPAMco-HC) occurs reversibly, regardless of the heating/cooling process. Figure 10A shows the change in fluorescence intensity, where the temperature is changed repeatedly between 25 and 40 °C. The data clearly show that the fluorescence intensity is reversibly changeable at least 10 times. Another notable feature of the polymer is the high reusability with a simple recovery process; heating the solution to 50 °C followed by centrifugation (5 min, 2.5 × 104 rpm) affords >98% polymer recovery. As shown in Figure 10B (white), the recovered polymer shows similar fluorescence response as does the virgin polymer (black), suggesting that the poly(NIPAM-co-HC) thermometer has high reusability.
4. Conclusion A new fluorescent thermometer, poly(NIPAM-co-HC), has been synthesized. The polymer shows very weak fluorescence at 25 °C, with a very high fluorescence enhancement factor (>20-fold). The fluorescence enhancement is driven by a heat-induced phase transition of the polymer. At low temperature, the HC unit exists as a nonfluorescent benzenoid form. However, at high temperature, the less polar domain formed within the aggregated polymer leads to transformation of the HC unit to the fluorescent quinoid form, resulting in strong fluorescence enhancement. The polymer can be used over a wide pH range (2-12) and is photoexcited by low-energy light (510 nm). In addition, the polymer shows high reusability and high reversibility of the fluorescence response. The polymer therefore has potential as a fluorescent thermometer with high sensitivity, reversibility, and reusability.
Figure 10. (A) Change in fluorescence intensity of poly(NIPAMco-HC) (0.1 g L-1) dissolved in water (pH 6.4), where the temperature was changed repeatedly between 25 and 40 °C. (B) Temperaturedependent change in fluorescence intensity (λexc ) 510 nm, λem ) 572 nm) of (black) virgin and (white) recovered poly(NIPAM-coHC) (0.1 g L-1) measured in water (pH 6.4) during a heating sequence. The recovery process of the polymer is heating the sample solution to 50 °C followed by centrifugation (5 min, 2.5 × 104 rpm). The gray symbol shows the data of the virgin polymer measured during a cooling sequence.
fluorescence intensity of poly(NIPAM-co-HC) even at acidic pH (Figure 9A) is therefore due to the deprotonation of the aniline
Acknowledgment. This work was supported by the Grantin-Aid for Scientific Research (No. 19760536) from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT). We thank Prof. T. Kitayama (Osaka University) for GPC analysis. We are also grateful to the Division of Chemical Engineering for the Lend-Lease Laboratory System. Supporting Information Available: Detailed fluorescence, absorption, and excitation spectra for the samples; Rh distribution data at different temperature; fluorescence and absorption spectra of 1; the temperature-dependent change in turbidity and hydrodynamic radius of the HC-free polyNIPAM; the change in the fluorescence intensity of 1 with pH; 1H, 13C NMR, and FAB-MS spectra of 1 and 2 (Figures S1S13). This material is available free of charge via the Internet at http://pubs.acs.org. LA703890N
