A fluorescent acrylamide-type monomer bearing an environment-sensitive methoxybenzocoumarin structure for the development of functional polymeric sensors†
Received
15th May 2016
, Accepted 11th August 2016
First published on 12th August 2016
Abstract
A new fluorescent acrylamide-type monomer bearing a hydrogen bonding- and polarity-sensitive benzocoumarin fluorophore was synthesized. The absorption spectra, fluorescence spectra, and fluorescence lifetime of a model compound were measured in ten solvents with different hydrogen-bonding abilities and polarities to investigate the sensitivity of the fluorophore to the surrounding environment. These spectroscopic studies demonstrated that the fluorophore emits stronger fluorescence in more protic, polar environments. A fluorescent polymeric thermometer was prepared from N-isopropylacrylamide and the new fluorescent monomer, and it showed good functionality in aqueous solution (e.g., high sensitivity to temperature changes and high chemical stability), indicating the applicability of the herein developed fluorescent monomer for use in functional sensors.
Introduction
Environment-sensitive fluorophores change their photophysical properties (e.g., fluorescence quantum yield, maximum emission wavelength, and fluorescence lifetime) depending on the surrounding environment. Fluorescent monomers consisting of an environment-sensitive fluorophore and a polymerizable vinyl bond are valuable in polymer chemistry because they can facilitate the elucidation of the microenvironments of macromolecules1 and the development of functional polymeric materials with applications in a wide range of scientific disciplines.2 Examples of the latter case include fluorescent monomers bearing an environment-sensitive dansyl,3 benzofurazan,4–6 aminocoumarin,7 or naphthalimide8 fluorophore, which have been used to construct fluorescent polymeric thermometers by us and other groups. Intracellular thermometry can now be performed with these fluorescent polymeric thermometers.6,9 Fluorescent polymeric sensors for potassium ions,10 sulfate ions,11 phosphatidylinositol-4,5-bisphosphate,12 and phosphatidylserine13 are also examples of macromolecular materials created from fluorescent monomers containing an environment-sensitive fluorophore. The use of some of these sensors12,13 has been explored to monitor biologically important molecules in live cell imaging. It should be noted that all of the fluorescent monomers in the functional polymers described above bear an environment-sensitive fluorophore, which shows stronger fluorescence in less protic, polar environments (e.g., in an aprotic organic solvent, such as ethyl acetate). Fluorescent monomers containing a fluorophore with the opposite sensitivity to the environment, i.e., those that emit stronger fluorescence in more protic, polar environments, are worthwhile components for the development of functional polymers with new properties.
In our previous study,14 we found that 8-methoxy-4-methyl-2H-benzo[g]chromen-2-one (MBC) (Fig. 1) is an environment-sensitive fluorophore that emits stronger fluorescence in more protic, polar environments. The unique fluorescent monomer (8-methoxy-2-oxo-2H-benzo[g]chromen-4-yl)methyl acrylate (MBC-AE), which bears the MBC structure, was synthesized, and a fluorescent polymeric thermometer, poly(N-isopropylacrylamide-co-MBC-AE) (poly(NIPAM-co-MBC-AE)) was prepared as an example of the use of this monomer. Poly(NIPAM-co-MBC-AE) showed temperature-dependent fluorescence in aqueous solution because the microenvironment near the MBC-AE units was changed by the thermo-responsive behavior of the polyNIPAM units.15 Nevertheless, the repeatable thermo-responsive function of poly(NIPAM-co-MBC-AE) was observed only under acidic conditions because the MBC-AE units in poly(NIPAM-co-MBC-AE) were hydrolyzed under neutral and basic conditions.
![image file: c6pp00150e-f1.tif](/image/article/2016/PP/c6pp00150e/c6pp00150e-f1.gif) |
| Fig. 1 Chemical structures of MBC, MBC-AE, poly(NIPAM-co-MBC-AE), MBC-AA, MBC-IA, and poly(NIPAM-co-MBC-AA). | |
To address this shortcoming, herein, we synthesized N-((8-methoxy-2-oxo-2H-benzo[g]chromen-4-yl)methyl)-N-methylacrylamide (MBC-AA) as a new acrylamide-type fluorescent monomer. The amide-type MBC-AA unit was expected to be more tolerant to hydrolysis than the ester-type MBC-AE unit when introduced into a polymer. N-((8-Methoxy-2-oxo-2H-benzo[g]chromen-4-yl)methyl)-N-methylisobutylamide (MBC-IA) was also synthesized as a model compound of the MBC-AA unit. The fluorescence properties of MBC-IA were investigated in ten solvents to determine the sensitivity of the fluorophore to the surrounding environment. In addition, poly(NIPAM-co-MBC-AA) was prepared, and its fluorescence properties were studied. The utility of the new fluorescent monomer MBC-AA was assessed by investigating the thermo-responsive function of poly(NIPAM-co-MBC-AA) in water.
Experimental
Materials and apparatus
Poly(NIPAM-co-MBC-AE) (weight-average molecular weight Mw = 148
000, number-average molecular weight Mn = 51
500, Mw/Mn = 2.87) was obtained as previously reported.141H NMR spectra were recorded using a Bruker Avance 400 spectrometer. The mass spectra acquired via electrospray ionization (ESI) were recorded using a Bruker micrOTOF-05 spectrometer. The melting points were measured using a Round Science RFS-10 and are uncorrected. The gel-permeation chromatography (GPC) equipment consisted of a JASCO PU-2080 pump, a JASCO RI-2031 refractive index detector, a JASCO FP-2020 fluorescence detector, a JASCO CO-2060 column thermostat, and a Shodex GPC KD-806 M column. A calibration curve was obtained using polystyrene standards, and 1-methyl-2-pyrrolidinone containing LiBr (5 mM) was used as the eluent.
Synthesis
8-Methoxy-4-((methylamino)methyl)-2H-benzo[g]chromen-2-one (MBC-MA).
4-Chloromethyl-8-methoxy-2H-benzo[g]chromen-2-one (100 mg, 0.36 mmol)14 was dissolved in acetonitrile (20 mL). After the addition of 40% methylamine solution (607 μL, 7.0 mmol), the mixture was stirred at 50 °C for 3 h. Then, the reaction mixture was evaporated to dryness under reduced pressure, and the residue was separated by chromatography on silica gel using dichloromethane–methanol (95
:
5, v/v) to afford MBC-MA (21.2 mg, 22%) as a pale yellow powder: mp, 132–133 °C; 1H NMR (CDCl3) δ 8.01 (1H, s), 7.76 (1H, d, J = 9.0 Hz), 7.51 (1H, s), 7.11 (1H, dd, J = 9.0, 2.2 Hz), 7.03 (1H, d, J = 2.2 Hz), 6.48 (1H, s), 4.00 (2H, s), 3.93 (3H, s), 2.58 (3H, s); 13C NMR (CDCl3) δ 161.8, 160.2, 153.6, 151.7, 136.9, 130.8, 126.2, 124.6, 120.1, 117.0, 113.6, 112.4, 105.1, 56.0, 52.2, 37.1. High-resolution ESI mass spectrometry (HR-ESI-MS): m/z Calcd for C16H16NO3+ [M + H]+ 270.1125. Found 270.1125.
MBC-AA
.
MBC-MA (70 mg, 0.26 mmol) was dissolved in acetonitrile (10 mL). After the addition of triethylamine (36.2 μL, 0.26 mmol) and acryloyl chloride (82.2 μL, 1.02 mmol), the mixture was stirred at 50 °C for 4.5 h. Then, Na2CO3 (1 g) was added to the solution to stop the reaction. After filtration to remove excess Na2CO3, the reaction mixture was evaporated to dryness under reduced pressure, and the residue was chromatographed on silica gel using dichloromethane–methanol (50
:
1, v/v) to afford MBC-AA (60.9 mg, 72%) as a yellow powder: mp, 166–167 °C; 1H NMR (CDCl3) δ 8.04, 7.91 (1H, s), 7.77 (1H, d, J = 9.2 Hz), 7.58, 7.55 (1H, s), 7.11–7.18 (1H, m), 7.07 (1H, s), 6.43–6.73 (2H, m), 6.24, 6.17 (1H, s), 5.69–5.84 (1H, m), 4.91, 4.87 (2H, s), 3.94 (3H, s), 3.16 (3H, s); 13C NMR (CDCl3) δ 166.7, 160.6, 159.7, 150.7, 150.0, 136.4, 130.3, 130.0, 129.5, 126.7, 125.5, 124.2, 123.0, 119.9, 119.5, 115.5, 112.6, 112.0, 111.7, 104.5, 55.4, 53.4, 50.1, 48.0, 35.7, 35.0, 29.6, 23.3. HR-ESI-MS: m/z Calcd for C19H17NNaO4+ [M + Na]+ 346.1050. Found 346.1055.
MBC-IA
.
MBC-MA (20 mg, 0.074 mmol) was dissolved in acetonitrile (3 mL). After the addition of triethylamine (13.5 μL, 0.097 mmol) and isobutyric anhydride (16.1 μL, 0.097 mmol), the mixture was stirred at room temperature for 4 h. Then K2CO3 (1 g) was added to the solution to stop the reaction. After filtration to remove excess K2CO3, the reaction mixture was evaporated to dryness under reduced pressure, and the residue was chromatographed on silica gel using dichloromethane–methanol (50
:
1, v/v) to afford MBC-IA (19.3 mg, 77%) as a yellow powder: mp, 188–190 °C; 1H NMR (CDCl3) δ 8.05, 7.93 (1H, s), 7.76–7.81 (1H, m), 7.64, 7.59 (1H, s), 7.09–7.18 (2H, m), 6.22, 6.17 (1H, s), 4.87, 4.84 (2H, s), 3.98, 3.95 (3H, s), 3.11, 3.09 (3H, s), 2.61–2.97 (m, 1H), 1.17–1.22 (6H, m); 13C NMR (CDCl3) δ 177.4, 160.8, 159.7, 150.9, 150.7, 136.5, 130.4, 125.6, 124.5, 119.6, 115.7, 113.1, 111.8, 104.5, 55.4, 47.9, 35.2, 30.5, 29.7, 19.2. HR-ESI-MS: m/z Calcd for C20H21NNaO4+ [M + Na]+ 362.1363. Found 362.1368.
Poly(NIPAM-co-MBC-AA).
NIPAM (2.5 mmol), MBC-AA (2.5 μmol), and α,α′-azobisisobutyronitrile (25 μmol) were dissolved in 1,4-dioxane (5 mL), and the solution was bubbled with dry Ar for 30 min to remove dissolved oxygen. The solution was heated at 60 °C for 6 h and then cooled to room temperature. The reaction mixture was then poured into diethyl ether (200 mL). The resulting polymer was collected by filtration and purified by reprecipitation using 1,4-dioxane (5 mL)–diethyl ether (100 mL) (yield: 76%). The proportion of the MBC-AA unit in the copolymer was determined by comparing the absorbance in methanol with that of the model fluorophore MBC-IA. The molecular weights (Mw and Mn) were determined by GPC with a refractive index detector.
Photophysical studies of MBC-IA
UV/Vis absorption spectra (10 or 30 μM) were recorded at 25 °C using a JASCO V-550 UV/Vis spectrophotometer. Fluorescence spectra (10 μM) were recorded using a JASCO FP-8500 spectrofluorimeter with a Hamamatsu R928 optional photomultiplier tube (operative range: 200–850 nm) at 25 °C and were corrected using a JASCO ESC-333 substandard light source. The fluorescence quantum yield of MBC-IA in ethanol (Φf = 0.26) was determined using a JASCO ILF-835 integrating sphere unit.6e The fluorescence quantum yields in other solvents were determined from eqn (1), where F is the area under the corrected fluorescence spectrum obtained with excitation at 345 nm, A is the absorbance at 345 nm, n is the refractive index of the solvent, and the subscripts R and S indicate the reference (i.e., MBC-IA in ethanol) and the sample, respectively. | Φf,s = Φf,RFSARnS2/FRASnR2 | (1) |
The fluorescence lifetimes (τf) were determined using a time-correlated single-photon counting (TCSPC) fluorimeter Horiba Jobin Yvon FluoroCube 3000U at 25 °C. The samples were excited with a Horiba NanoLED-370 (excitation: 370 nm) at a repetition rate of 1 MHz. The recorded fluorescence decay curves (I(t)) were fitted by an exponential function expressed as eqn (2), where B is the pre-exponential factor, and t is the time.
| ![image file: c6pp00150e-t1.tif](/image/article/2016/PP/c6pp00150e/c6pp00150e-t1.gif) | (2) |
Then, the τf values were calculated using eqn (3).
| ![image file: c6pp00150e-t2.tif](/image/article/2016/PP/c6pp00150e/c6pp00150e-t2.gif) | (3) |
The fractional contributions (Pi for τi) were calculated using eqn (4).
| ![image file: c6pp00150e-t3.tif](/image/article/2016/PP/c6pp00150e/c6pp00150e-t3.gif) | (4) |
According to the fluorescence lifetime and the fluorescence quantum yield, the fluorescence rate constant (kf) and non-radiative rate constant (knr) were calculated using eqn (5) and (6), respectively.
| ![image file: c6pp00150e-t4.tif](/image/article/2016/PP/c6pp00150e/c6pp00150e-t4.gif) | (5) |
| ![image file: c6pp00150e-t5.tif](/image/article/2016/PP/c6pp00150e/c6pp00150e-t5.gif) | (6) |
Functional studies of poly(NIPAM-co-MBC-AA)
The fluorescence spectra of poly(NIPAM-co-MBC-AA) (0.01% w/v) were recorded in 1,4-dioxane, acetonitrile, ethanol, methanol, and water with excitation at 345 nm. After ten cycles of heating (to 45 °C) and cooling (to 25 °C) in water, the polymer solution was lyophilized, and the residue was re-dissolved in 1-methyl-2-pyrrolidinone (0.5% w/v) for subsequent GPC. The hydrodynamic diameter of poly(NIPAM-co-MBC-AA) was estimated from dynamic light scattering (DLS) measurements using a Zetasizer Nano ZS (Malvern Instruments). The samples (0.01% w/v) were equilibrated at 20 °C for 10 min.
Results and discussion
Synthesis of MBC-AA and MBC-IA
The new fluorescent monomer MBC-AA and the model compound MBC-IA were synthesized from 4-chloromethyl-8-methoxy-2H-benzo[g]chromen-2-one14 by two-step reactions (Fig. 2). In the first reaction, the methylamino group was introduced by nucleophilic substitution under mild conditions. Then, acylation was performed using acryloyl chloride and isobutyric anhydride to obtain MBC-AA and MBC-IA, respectively.
![image file: c6pp00150e-f2.tif](/image/article/2016/PP/c6pp00150e/c6pp00150e-f2.gif) |
| Fig. 2 Synthesis of MBC-AA and MBC-IA. (i) 40% MeNH2, MeCN, 50 °C, 3 h (22%); (ii) acryloyl chloride, Et3N, MeCN, 50 °C, 4.5 h (72%); and (iii) isobutyric anhydride, Et3N, MeCN, rt, 4 h (77%). | |
Fluorescence properties of MBC-IA
The absorption and fluorescence spectra of the model fluorophore MBC-IA were recorded in ten solvents (n-hexane, 1,4-dioxane, ethyl acetate, acetonitrile, chloroform, ethanol, methanol, a mixture of water and methanol [4
:
1, v/v], water, and trifluoroethanol) (Fig. 3). Table 1 summarizes the photophysical properties of MBC-IA in these solvents and the solvents’ hydrogen-bonding ability (i.e., hydrogen-bond donor acidity α (ref. 16)) and polarity (i.e., dielectric constant D (ref. 17)). Similar to MBC,14 although MBC-IA was almost non-fluorescent in aprotic, apolar solvents (e.g., Φf = 0.0054 in n-hexane), it strongly fluoresced in protic, polar solvents (e.g., Φf = 0.35 in methanol).
![image file: c6pp00150e-f3.tif](/image/article/2016/PP/c6pp00150e/c6pp00150e-f3.gif) |
| Fig. 3 Absorption (black line) and fluorescence (red line) spectra of MBC-IA (10 μM) in various solvents at 25 °C. All fluorescence spectra were recorded with excitation at the maximum absorption wavelength. | |
Table 1 Photophysical properties of MBC-IA: fluorescence quantum yield (Φf), maximum absorption wavelength (λabs), molar absorption coefficient (ε), maximum emission wavelength (λem), fluorescence lifetime (τf), fluorescence rate constant (kf), and non-radiative rate constant (knr)
Solvent |
α
|
D
|
Φ
f
|
λ
abs/nm |
ε/M−1 cm−1 |
λ
em/nm |
τ
f/ns |
k
f/107 s−1 |
k
nr/107 s−1 |
Hydrogen-bond donor acidity of the solvent.16
Dielectric constant of the solvent.17
Average fluorescence lifetime of three components: τ1 = 0.21 ns (45%), τ2 = 1.1 ns (51%), and τ3 = 5.0 ns (3.7%).
Average fluorescence lifetime of two components: τ1 = 0.63 ns (53%) and τ2 = 5.1 ns (47%).
Average fluorescence lifetime of two components: τ1 = 1.2 ns (69%) and τ2 = 7.0 ns (31%).
|
n-Hexane |
0.00 |
1.9 |
0.0054 |
342 |
16 100 |
425 |
0.98 |
0.55 |
100 |
1,4-Dioxane |
0.00 |
2.2 |
0.0033 |
343 |
16 300 |
462 |
0.86c |
0.38 |
116 |
Ethyl acetate |
0.00 |
6.0 |
0.0053 |
342 |
15 500 |
432 |
1.1 |
0.47 |
87 |
Acetonitrile |
0.19 |
37.5 |
0.031 |
343 |
15 200 |
483 |
2.7d |
1.1 |
35 |
Chloroform |
0.44 |
4.8 |
0.052 |
349 |
16 400 |
468 |
3.0e |
1.7 |
32 |
Ethanol |
0.83 |
24.3 |
0.26 |
347 |
15 600 |
500 |
5.2 |
4.9 |
14 |
Methanol |
0.93 |
32.6 |
0.35 |
346 |
14 900 |
512 |
6.3 |
5.5 |
10 |
Water–methanol [4 : 1, v/v] |
|
70.7 |
0.28 |
348 |
14 400 |
540 |
4.4 |
6.3 |
16 |
Water |
1.17 |
78.5 |
0.17 |
346 |
13 100 |
540 |
3.4 |
5.1 |
24 |
Trifluoroethanol |
1.51 |
26.8 |
0.33 |
349 |
15 600 |
538 |
6.0 |
5.4 |
11 |
Regarding the maximum emission wavelength, MBC-IA fluoresced at longer wavelengths as the hydrogen-bonding ability and polarity of the solvent increased, indicating that the first singlet state of MBC-IA has an intramolecular charge transfer (ICT) character.18Table 1 also shows the fluorescence lifetimes (see Fig. S1† for the original fluorescence decay curves), fluorescence rate constants, and non-radiative rate constants of MBC-IA in the different solvents. The high efficiency of the non-radiative relaxation process in MBC-IA in aprotic, apolar solvents is because of the proximity of an emissive ππ* state to a non-emissive nπ* state.19 Comparing the photophysical properties of MBC-IA in acetonitrile (Φf = 0.031, hydrogen-bond donor acidity α = 0.19 and dielectric constant D = 37.5) and in methanol (Φf = 0.35, α = 0.93, and D = 32.6) revealed that the fluorescence process of MBC-IA is dominantly influenced by the hydrogen-bonding ability of the solvent rather than by its polarity. The non-radiative process of MBC-IA in water was accelerated compared to that in methanol, although water has a stronger hydrogen-bonding ability (α = 1.17) than methanol (α = 0.93). The proximity of the first singlet ππ* state to the ground state in more polar environments, as indicated by the longer emission wavelength (i.e., 540 nm in water), increases the efficiency of the non-radiative internal conversion. This effect has been established as the “energy gap law”20 in photochemistry. It should be also noted that the fluorescence rate constant of MBC-IA was strongly influenced by the solvents (see Table 1). Because such a decrease in the kf value in the non-polar solvents (i.e., n-hexane, 1,4-dioxane and ethyl acetate) was not observed in the original fluorophore MBC,19 the dependency of the kf values of MBC-IA on the solvents can be ascribed to the interaction of the fluorophore (i.e., the methoxybenzocoumarin moiety) with the side chain (i.e., the methylisobutylamide moiety) in the non-polar solvents.
To further demonstrate the sensitivity of MBC-IA to the surrounding environment, its fluorescence spectra were obtained in mixtures of aprotic and apolar 1,4-dioxane (α = 0.00, and D = 2.2) and protic and polar trifluoroethanol (α = 1.51, and D = 26.8). As shown in Fig. 4, the fluorescence intensity of MBC-IA dramatically increased as the molar ratio of trifluoroethanol in the mixture increased. These results are in accordance with the general observation that MBC-IA emits more strongly in more protic, polar media.
![image file: c6pp00150e-f4.tif](/image/article/2016/PP/c6pp00150e/c6pp00150e-f4.gif) |
| Fig. 4 Fluorescence spectra of MBC-IA (10 μM) in a mixture of 1,4-dioxane and trifluoroethanol at 25 °C. All samples were excited at 345 nm. | |
Fluorescence properties of poly(NIPAM-co-MBC-AA)
Next, poly(NIPAM-co-MBC-AA) (m
:
n = 100
:
0.06 in Fig. 1, Mw = 125
000, Mn = 41
000, and Mw/Mn = 3.04) was prepared by random copolymerization, and its fluorescence properties were investigated. First, the fluorescence spectra of poly(NIPAM-co-MBC-AA) were obtained in organic solvents (1,4-dioxane, acetonitrile, ethanol, and methanol). In these solvents, poly(NIPAM-co-MBC-AA) takes a flexible form, and the hydrodynamic diameters of poly(NIPAM-co-MBC-AA) at 20 °C were estimated by DLS measurements to be 10.35 ± 0.36, 8.00 ± 1.24, 15.95 ± 0.84, and 10.22 ± 1.70 nm in 1,4-dioxane, acetonitrile, ethanol, and methanol, respectively (Fig. S2†). As shown in Fig. 5, poly(NIPAM-co-MBC-AA) emitted stronger fluorescence at longer wavelengths in a more protic, polar solvent. Thus, the sensitivity of the methoxybenzocoumarin fluorophore (i.e., MBC) to the surrounding environment was preserved, even when it was introduced into a polymer structure.
![image file: c6pp00150e-f5.tif](/image/article/2016/PP/c6pp00150e/c6pp00150e-f5.gif) |
| Fig. 5 Fluorescence spectra of poly(NIPAM-co-MBC-AA) in 1,4-dioxane, acetonitrile, ethanol, and methanol at 25 °C. All samples (0.01% w/v) were excited at 345 nm. | |
Finally, the fluorescence properties of poly(NIPAM-co-MBC-AA) in water (hydrodynamic diameter: 12.40 ± 1.55 nm at 20 °C, Fig. S2†) were evaluated by changing the temperature. Fig. 6a and b show the fluorescence responses of poly(NIPAM-co-MBC-AA) to increasing temperatures. This fluorescence behavior is attributable to change in the microenvironment of the NIPAM units that occurs at approximately 32 °C: solvent water molecules are repelled from the NIPAM units at temperatures higher than 32 °C.15 As displayed in Fig. 6c, the fluorescence response of poly(NIPAM-co-MBC-AA) to the temperature variation was highly reproducible over ten cycles of heating and cooling, whereas that of poly(NIPAM-co-MBC-AE) lacked stability and sensitivity. The difference in the functional reproducibility between poly(NIPAM-co-MBC-AA) and poly(NIPAM-co-MBC-AE) was attributed to the robustness of the fluorescent units. Aliquots of aqueous poly(NIPAM-co-MBC-AA) and poly(NIPAM-co-MBC-AE) after ten cycles of functional assessment (heating to 45 °C and cooling to 25 °C) were analyzed by GPC with a fluorescence detector. As indicated in Fig. 7, the MBC-AA units of poly(NIPAM-co-MBC-AA) remained unchanged during the heating and cooling cycles, whereas the MBC-AE units of poly(NIPAM-co-MBC-AE) decomposed significantly. The ester linkage of MBC-AE units was easily hydrolyzed, even in neutral aqueous solution. The fluorophore cleaved from poly(NIPAM-co-MBC-AE) was no longer sensitive to the heat-induced local environmental change near the NIPAM units, resulting in the deterioration of the function as seen in Fig. 6c. On the other hand, the new fluorescent monomer MBC-AA does not exhibit the low stability of the conventional monomer MBC-AE.
![image file: c6pp00150e-f6.tif](/image/article/2016/PP/c6pp00150e/c6pp00150e-f6.gif) |
| Fig. 6 Function of poly(NIPAM-co-MBC-AA) in water as a fluorescent polymeric thermometer. (a) Representative fluorescence spectra with increasing temperature. In the region indicated by dotted lines near 690 nm, scatter due to excitation light overlapped with the fluorescence spectra. (b) Change in the fluorescence intensity at 520 nm. Normalized at 25 °C. (c) Reversibility of the fluorescence intensities of poly(NIPAM-co-MBC-AA) (left) and a reference copolymer poly(NIPAM-co-MBC-AE) (right) over ten cycles of heating (45 °C, open circle) and cooling (25 °C, closed circle). (d) Change in the fluorescence intensity ratio at 470 nm and 520 nm (FI470/FI520). (e) Reversibility of the fluorescence ratio FI470/FI520 over ten cycles of heating (45 °C, open circle) and cooling (25 °C, closed circle). The data shown in (b)–(e) were obtained from the fluorescence spectra collected as the temperature increased. The relative standard deviations (RSDs) indicated in (c) and (e) were calculated from the fluorescence intensities at each temperature (n = 10). All samples (0.01% w/v) were excited at 345 nm. | |
![image file: c6pp00150e-f7.tif](/image/article/2016/PP/c6pp00150e/c6pp00150e-f7.gif) |
| Fig. 7 Stability of poly(NIPAM-co-MBC-AA) in water. Chromatograms of pristine poly(NIPAM-co-MBC-AA) (a) and poly(NIPAM-co-MBC-AE) (c), and the corresponding samples after ten cycles of heating (45 °C) and cooling (25 °C) in water (b and d, respectively). Peaks: 1, poly(NIPAM-co-MBC-AA); 2, poly(NIPAM-co-MBC-AE); and 3, fluorescent compounds cleaved from poly(NIPAM-co-MBC-AE). The fluorescence intensity at 470 nm was monitored with excitation at 345 nm. | |
In the fluorescence response of poly(NIPAM-co-MBC-AA) in water (Fig. 6a), the maximum emission wavelength shifted from 520 to 470 nm as the temperature increased from 30 to 40 °C. This remarkable spectral shift enabled us to consider using poly(NIPAM-co-MBC-AA) as a ratiometric fluorescent thermometer. Fig. 6d shows the relationship between the fluorescence intensity ratio of poly(NIPAM-co-MBC-AA) at 470 and 520 nm (FI470/FI520) and the temperature. The FI470/FI520 value was also highly reproducible over ten cycles of heating and cooling (Fig. 6e). Although the functional temperature range was relatively narrow, the average sensitivity of the fluorescence intensity ratio (FI470/FI520) of poly(NIPAM-co-MBC-AA) as the temperature varied from 32 to 38 °C was 15.0% °C−1, which is much higher than those of the conventional ratiometric fluorescent thermometers functioning in water, such as fluorophore(s)-labeled thermo-responsive polymers (3.4–11.3% °C−1),4a,6e,21 fluorophore(s)- and/or lumophore(s)-containing polymer nanoparticles (2.0–4.2% °C−1),22 emissive inorganic nanoparticles (0.26–3.1% °C−1),23 a small organic molecule (2.7% °C−1),24 GFP (1.3% °C−1),25 and others (1.7–3.9% °C−1)26 (see Table S1† for a detailed comparison). The high sensitivity of poly(NIPAM-co-MBC-AA) clearly originated from the environment-sensitive MBC-AA units. Furthermore, poly(NIPAM-co-MBC-AA) has several advantages in ratiometric temperature sensing because (a) it requires only a single excitation to exhibit fluorescence at two different wavelengths; (b) the MBC-AA unit is pH insensitive because of the lack of a pH-sensitive structure, such as an amino group or a phenolic hydroxyl group; and (c) it contains only one fluorophore, which reduces the possibility of undesirable interactions with bio-relevant molecules when applied to probe cellular temperatures. In summary, the new fluorescent monomer MBC-AA is expected to aid in the development of novel functional polymeric sensors, especially because MBC-AA is highly robust and highly sensitive to environmental changes. In the near future, new stimulus-responsive polymeric materials will be created through the application of MBC-AA. Our research group is now pursuing this research direction.
Acknowledgements
We acknowledge the Japan Science and Technology Agency (the Development of Advanced Measurement and Analysis Systems program for S. U.), the Japan Society for the Promotion of Science (Grant-in-Aid for Challenging Exploratory Research 16K14002 for S. U.), and the Spanish Ministry for Economy and Competitiveness (grants CTQ2008-06777-C02-02 and CTQ2014-54729-C2-1-P for U. P. and PhD fellowship BES-2009-012264 and travel grant EEBB-I-13-07155 for P. R.).
Notes and references
-
(a) K. J. Shea, G. J. Stoddard, D. M. Shavelle, F. Wakui and R. M. Choate, Synthesis and characterization of highly cross-linked polyacrylamides and polymethacrylamides. A new class of macroporous polyamides, Macromolecules, 1990, 23, 4497–4507 CrossRef CAS;
(b) Y. Hattori, K. Nagase, J. Kobayashi, A. Kikuchi, Y. Akiyama, H. Kanazawa and T. Okano, Hydration of poly(N-isopropylacrylamide) brushes on micro-silica beads measured by a fluorescent probe, Chem. Phys. Lett., 2010, 491, 193–198 CrossRef CAS.
-
(a) Z. Yang, J. Cao, Y. He, J. H. Yang, T. Kim, X. Peng and J. S. Kim, Macro-/micro-environment-sensitive chemosensing and biological imaging, Chem. Soc. Rev., 2014, 43, 4563–4601 RSC;
(b) J. Hu and S. Liu, Responsive polymers for detection and sensing applications: current status and future developments, Macromolecules, 2010, 43, 8315–8330 CrossRef CAS.
- A. Yamada, Y. Hiruta, J. Wang, E. Ayano and H. Kanazawa, Design of environmentally responsive fluorescent polymer probes for cellular imaging, Biomacromolecules, 2015, 16, 2356–2362 CrossRef CAS PubMed.
-
(a) T. Wu, G. Zou, J. Hu and S. Liu, Fabrication of photoswitchable and thermotunable multicolor fluorescent hybrid silica nanoparticles coated with dye-labeled poly(N-isopropylacrylamide) brushes, Chem. Mater., 2009, 21, 3788–3798 CrossRef CAS;
(b) C. Li, Y. Zhang, J. Hu, J. Cheng and S. Liu, Reversible three-state switching of multicolor fluorescence emission by multiple stimuli modulated FRET processes within thermoresponsive polymeric micelles, Angew. Chem., Int. Ed., 2010, 49, 5120–5124 CrossRef CAS PubMed;
(c) Y. Wu, H. Hu, J. Hu, T. Liu, G. Zhang and S. Liu, Thermo- and light-regulated formation and disintegration of double hydrophilic block copolymer assemblies with tunable fluorescence emissions, Langmuir, 2013, 29, 3711–3720 CrossRef CAS PubMed.
-
(a) S. Uchiyama, Y. Matsumura, A. P. de Silva and K. Iwai, Fluorescent molecular thermometers based on polymers showing temperature-induced phase transitions and labeled with polarity-responsive benzofurazans, Anal. Chem., 2003, 75, 5926–5935 CrossRef CAS PubMed;
(b) S. Uchiyama, Y. Matsumura, A. P. de Silva and K. Iwai, Modulation of the sensitive temperature range of fluorescent molecular thermometers based on thermoresponsive polymers, Anal. Chem., 2004, 76, 1793–1798 CrossRef CAS PubMed;
(c) S. Uchiyama, N. Kawai, A. P. de Silva and K. Iwai, Fluorescent polymeric AND logic gate with temperature and pH as inputs, J. Am. Chem. Soc., 2004, 126, 3032–3033 CrossRef CAS PubMed;
(d) C. Gota, S. Uchiyama and T. Ohwada, Accurate fluorescent polymeric thermometers containing an ionic component, Analyst, 2007, 132, 121–126 RSC;
(e) C. Gota, S. Uchiyama, T. Yoshihara, S. Tobita and T. Ohwada, Temperature-dependent fluorescence lifetime of a fluorescent polymeric thermometer, poly(N-isopropylacrylamide), labeled by polarity and hydrogen bonding sensitive 4-sulfamoyl-7-aminobenzofurazan, J. Phys. Chem. B, 2008, 112, 2829–2836 CrossRef CAS PubMed.
-
(a) C. Gota, K. Okabe, T. Funatsu, Y. Harada and S. Uchiyama, Hydrophilic fluorescent nanogel thermometer for intracellular thermometry, J. Am. Chem. Soc., 2009, 131, 2766–2767 CrossRef CAS PubMed;
(b) K. Okabe, N. Inada, C. Gota, Y. Harada, T. Funatsu and S. Uchiyama, Intracellular temperature mapping with a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy, Nat. Commun., 2012, 3, 705 CrossRef PubMed;
(c) T. Tsuji, S. Yoshida, A. Yoshida and S. Uchiyama, Cationic fluorescent polymeric thermometers with the ability to enter yeast and mammalian cells for practical intracellular temperature measurements, Anal. Chem., 2013, 85, 9815–9823 CrossRef CAS PubMed;
(d) T. Hayashi, N. Fukuda, S. Uchiyama and N. Inada, A cell-permeable fluorescent polymeric thermometer for intracellular temperature mapping in mammalian cell lines, PLoS One, 2015, 10, e0117677 Search PubMed;
(e) S. Uchiyama, T. Tsuji, K. Ikado, A. Yoshida, K. Kawamoto, T. Hayashi and N. Inada, A cationic fluorescent polymeric thermometer for the ratiometric sensing of intracellular temperature, Analyst, 2015, 140, 4498–4506 RSC.
- S. Inal, J. D. Kölsch, F. Sellrie, J. A. Schenk, E. Wischerhoff, A. Laschewsky and D. Neher, A water soluble fluorescent polymer as a dual colour sensor for temperature and a specific protein, J. Mater. Chem. B, 2013, 1, 6373–6381 RSC.
- S. Inal, J. D. Kölsch, L. Chiappisi, D. Janietz, M. Gradzielski, A. Laschewsky and D. Neher, Structure-related differences in the temperature-regulated fluorescence response of LCST type polymers, J. Mater. Chem. C, 2013, 1, 6603–6612 RSC.
-
(a) N. Inada and S. Uchiyama, Methods and benefits of imaging the temperature distribution inside living cells, Imaging Med., 2013, 5, 303–305 CrossRef CAS;
(b)
S. Uchiyama and N. Inada, Cellular thermometry, in Thermometry at the Nanoscale: Techniques and Selected Applications, ed. L. D. Carlos and F. Palacio, Royal Society of Chemistry, Cambridge, 2016, pp. 355–382 Search PubMed.
- J. Yin, C. Li, D. Wang and S. Liu, FRET-derived ratiometric fluorescent K+ sensors fabricated from thermoresponsive poly(N-isopropylacrylamide) microgels labeled with crown ether moieties, J. Phys. Chem. B, 2010, 114, 12213–12220 CrossRef CAS PubMed.
- M. Onoda, S. Uchiyama and T. Ohwada, Fluorogenic ion sensing system working in water, based on stimulus-responsive copolymers incorporating a polarity-sensitive fluorophore, Macromolecules, 2007, 40, 9651–9657 CrossRef CAS.
- Y. Yoon, P. J. Lee, S. Kurilova and W. Cho,
In situ quantitative imaging of cellular lipids using molecular sensors, Nat. Chem., 2011, 3, 868–874 CrossRef CAS PubMed.
- S.-L. Liu, R. Sheng, M. J. O'Connor, Y. Cui, Y. Yoon, S. Kurilova, D. Lee and W. Cho, Simultaneous in situ quantification of two cellular lipid pools using orthogonal fluorescent sensors, Angew. Chem., Int. Ed., 2014, 53, 14387–14391 CrossRef CAS PubMed.
- S. Uchiyama, K. Takehira, T. Yoshihara, S. Tobita and T. Ohwada, Environment-sensitive fluorophore emitting in protic environments, Org. Lett., 2006, 8, 5869–5872 CrossRef CAS PubMed.
-
(a) H. G. Schild, Poly(N-isopropylacrylamide): experiment, theory and application, Prog. Polym. Sci., 1992, 17, 163–249 CrossRef CAS;
(b) A. Halperin, M. Kröger and F. M. Winnik, Poly(N-isopropylacrylamide) phase diagrams: fifty years of research, Angew. Chem., Int. Ed., 2015, 54, 15342–15367 CrossRef CAS PubMed.
- M. J. Kamlet, J.-L. M. Abboud, M. H. Abraham and R. W. Taft, Linear solvation energy relationships. 23. A comprehensive collection of the solvatochromic parameters, π*, α, and β, and some methods for simplifying the generalized solvatochromic equation, J. Org. Chem., 1983, 48, 2877–2887 CrossRef CAS.
-
(a)
A. A. Maryott and E. R. Smith, Table of Dielectric Constants of Pure Liquids, U. S. Govt. Print. Off., Washington, 1951 Search PubMed;
(b) T. Shedlovsky and R. L. Kay, The ionization constant of acetic acid in water-methanol mixtures at 25° from conductance measurements, J. Phys. Chem., 1956, 60, 151–155 CrossRef CAS;
(c) Y. Tanaka, Y. F. Xiao and S. Matsuo, Relative permittivity of fluoroalcohols at temperature from 293 to 323 K and pressures up to 50 MPa, Fluid Phase Equilib., 2000, 170, 139–149 CrossRef CAS.
-
(a)
J. R. Lakowicz, in Principles of Fluorescence Spectroscopy, Springer, New York, 3rd edn, 2006, p. 205 Search PubMed;
(b)
B. Valeur and M. N. Berberan-Santos, in Molecular
Fluorescence: Principles and Applications, Wiley, Weinheim, 2nd edn, 2012, p. 379 Search PubMed.
- A. Kobayashi, K. Takehira, T. Yoshihara, S. Uchiyama and S. Tobita, Remarkable fluorescence enhancement of benzo[g]chromen-2-ones induced by hydrogen-bonding interactions with protic solvents, Photochem. Photobiol. Sci., 2012, 11, 1368–1376 CAS.
-
(a) R. Englman and J. Jortner, The energy gap law for non-radiative decay in large molecules, J. Lumin., 1970, 1–2, 134–142 CrossRef;
(b)
N. J. Turro, in Modern Molecular Photochemistry, University Science Books, Sausalito, 1991, p. 183 Search PubMed.
-
(a) C. Pietsch, A. Vollrath, R. Hoogenboom and U. S. Schubert, A fluorescent thermometer based on a pyrene-labeled thermoresponsive polymer, Sensors, 2010, 10, 7979–7990 CrossRef CAS PubMed;
(b) C.-Y. Chen and S.-T. Chen, A PNIPAM-based fluorescent nanothermometer with ratiometric readout, Chem. Commun., 2011, 47, 994–996 RSC;
(c) J. Hu, L. Dai and S. Liu, Analyte-reactive amphiphilic thermoresponsive diblock copolymer micelles-based multifunctional ratiometric fluorescent chemosensors, Macromolecules, 2011, 44, 4699–4710 CrossRef CAS;
(d) J. Hu, X. Zhang, D. Wang, X. Hu, T. Liu, G. Zhang and S. Liu, Ultrasensitive ratiometric fluorescent pH and temperature probes constructed from dye-labeled thermoresponsive double hydrophilic block copolymers, J. Mater. Chem., 2011, 21, 19030–19038 RSC;
(e) J. Liu, X. Guo, R. Hu, J. Xu, S. Wang, S. Li, Y. Li and G. Yang, Intracellular fluorescent temperature probe based on triarylboron substituted poly N-isopropylacrylamide and energy transfer, Anal. Chem., 2015, 87, 3694–3698 CrossRef CAS PubMed;
(f) J. Qiao, Y.-H. Hwang, C.-F. Chen, L. Qi, P. Dong, X.-Y. Mu and D.-P. Kim, Ratiometric fluorescent polymeric thermometer for thermogenesis investigation in living cells, Anal. Chem., 2015, 87, 10535–10541 CrossRef CAS PubMed;
(g) F. Pinaud, R. Millereux, P. Vialar-Trarieux, B. Catargi, S. Pinet, I. Gosse, N. Sojic and V. Ravaine, Differential photoluminescent and electrochemiluminescent behavior for resonance energy transfer processes in thermoresponsive microgels, J. Phys. Chem. B, 2015, 119, 12954–12961 CrossRef CAS PubMed.
-
(a) Y. Takei, S. Arai, A. Murata, M. Takabayashi, K. Oyama, S. Ishiwata, S. Takeoka and M. Suzuki, A nanoparticle-based ratiometric and self-calibrated fluorescent thermometer for single living cells, ACS Nano, 2014, 8, 198–206 CrossRef CAS PubMed;
(b) S. Arai, Ferdinandus, S. Takeoka, S. Ishiwata, H. Sato and M. Suzuki, Micro-thermography in millimeter-scale animals by using orally-dosed fluorescent nanoparticle thermosensors, Analyst, 2015, 140, 7534–7539 RSC;
(c) R. Piñol, C. D. S. Brites, R. Bustamante, A. Martínez, N. J. O. Silva, J. L. Murillo, R. Cases, J. Carrey, C. Estepa, C. Sosa, F. Palacio, L. D. Carlos and A. Millán, Joining time-resolved thermometry and magnetic-induced heating in a single nanoparticle unveils intriguing thermal properties, ACS Nano, 2015, 9, 3134–3142 CrossRef PubMed.
-
(a) F. Vetrone, R. Naccache, A. Zamarrón, A. J. de la Fuente, F. Sanz-Rodríguez, L. M. Maestro, E. M. Rodriguez, D. Jaque, J. G. Solé and J. A. Capobianco, Temperature sensing using fluorescent nanothermometers, ACS Nano, 2010, 4, 3254–3258 CrossRef CAS PubMed;
(b) E. J. McLaurin, V. A. Vlaskin and D. R. Gamelin, Water-soluble dual-emitting nanocrystals for ratiometric optical
thermometry, J. Am. Chem. Soc., 2011, 133, 14978–14980 CrossRef CAS PubMed;
(c) N.-N. Dong, M. Pedroni, F. Piccinelli, G. Conti, A. Sbarbati, J. E. Ramírez-Hernández, L. M. Maestro, M. C. Iglesias-de la Cruz, F. Sanz-Rodriguez, A. Juarranz, F. Chen, F. Vetrone, J. A. Capobianco, J. G. Solé, M. Bettinelli, D. Jaque and A. Speghini, NIR-to-NIR two-photon excited CaF2: Tm3+, Yb3+ nanoparticles: multifunctional nanoprobes for highly penetrating fluorescence bio-imaging, ACS Nano, 2011, 5, 8665–8671 CrossRef CAS PubMed;
(d) A. Sedlmeier, D. E. Achatz, L. H. Fischer, H. H. Gorris and O. S. Wolfbeis, Photon upconverting nanoparticles for luminescent sensing of temperature, Nanoscale, 2012, 4, 7090–7096 RSC;
(e) R. Tanimoto, T. Hiraiwa, Y. Nakai, Y. Shindo, K. Oka, N. Hiroi and A. Funahashi, Detection of temperature difference in neuronal cells, Sci. Rep., 2016, 6, 22071 CrossRef CAS PubMed.
- M. Homma, Y. Takei, A. Murata, T. Inoue and S. Takeoka, A ratiometric fluorescent molecular probe for visualization of mitochondrial temperature in living cells, Chem. Commun., 2015, 51, 6194–6197 RSC.
- S. Kiyonaka, T. Kajimoto, R. Sakaguchi, D. Shinmi, M. Omatsu-Kanbe, H. Matsuura, H. Imamura, T. Yoshizaki, I. Hamachi, T. Morii and Y. Mori, Genetically encoded fluorescent thermosensors visualize subcellular thermoregulation in living cells, Nat. Methods, 2013, 10, 1232–1238 CrossRef CAS PubMed.
-
(a) H.-S. Peng, S.-H. Huang and O. S. Wolfbeis, Ratiometric fluorescent nanoparticles for sensing temperature, J. Nanopart. Res., 2010, 12, 2729–2733 CrossRef CAS;
(b) F. Ye, C. Wu, Y. Jin, Y.-H. Chan, X. Zhang and D. T. Chiu, Ratiometric temperature sensing with semiconducting polymer dots, J. Am. Chem. Soc., 2011, 133, 8146–8149 CrossRef CAS PubMed;
(c) A. E. Albers, E. M. Chan, P. M. McBride, C. M. Ajo-Franklin, B. E. Cohen and B. A. Helms, Dual-emitting quantum dot/quantum rod-based nanothermometers with enhanced response and sensitivity in live cells, J. Am. Chem. Soc., 2012, 134, 9565–9568 CrossRef CAS PubMed.
Footnote |
† Electronic supplementary information (ESI) available: Table S1 and Fig. S1, S2. See DOI: 10.1039/c6pp00150e |
|
This journal is © The Royal Society of Chemistry and Owner Societies 2016 |