Fluorescent, stimuli-responsive, crosslinked PNIPAM-based microgel

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Fluorescent, stimuli-responsive, crosslinked PNIPAM-based microgel Yongkyun Kim, Daigeun Kim, Geunseok Jang, Jongho Kim, Taek Seung Lee ∗ Organic and Optoelectronic Materials Laboratory, Department of Advanced Organic Materials and Textile System Engineering, Chungnam National University, Daejeon 305-764, Republic of Korea

a r t i c l e

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Article history: Received 23 July 2014 Received in revised form 15 October 2014 Accepted 20 October 2014 Available online xxx Keywords: Stimuli-responsive material PNIPAM microgel Sensor pH and temperature

a b s t r a c t We have synthesized a highly pH and temperature-sensitive, fluorescent, crosslinked poly(Nisopropylacrylamide) (PNIPAM)-based probe via simple free radical emulsion copolymerization, which formed a reversible microgel containing both fluorophores of fluorescein and rhodamine derivatives. We confirmed that the opening of the lactone ring of fluorescein and the lactam ring of rhodamine in the polymer was responsible for the fluorescence changes in basic and acidic conditions, which showed fluorescence emissions at 514 nm (green) and 586 nm (red), respectively. The polymeric microgel exhibited a linear relationship between the ratio of emission intensity and pH windows of pH 2–6 and 7–12. Moreover, the microgel exhibited changes in fluorescence intensity with an alteration of turbidity of the microgel induced by temperature. Moreover, these fluorescence changes could be easily observed by the naked eye, serving as a potential probe for ratiometric microgel detection of pH or temperature. © 2014 Elsevier B.V. All rights reserved.

1. Introduction During the past few years, much attention has been paid to stimuli-responsive polymeric materials for potential applications [1–3]. For example, they typically undergo reversible or irreversible changes in physical properties and chemical structures as the result of external stimuli such as pH, temperature, light irradiation, ionic strength, and electric and magnetic fields. Thus, such techniques have been used to deliver useful drugs to targeted areas using stimuli-responsive carriers, such as imaging of hypoxic cells at low pH, low oxygen partial pressure, and a high level of bioreductive molecules [4–7]. A variety of nanoparticles have been used in enzyme activity, cell imaging, phototherapy, and intracellular application. To do these applications, various shapes of nanoparticles such as gold, polypyrrole particles, hydrogels, quantum dots, and polymer nanoparticles have been employed [8–12]. Micro-sized PNIPAM hydrogels have been widely studied in applications to drug delivery, tissue engineering, and responsive colloidal arrays during recent decades. Above a temperature commonly referred to as the lower critical solution temperature (LCST), PNIPAM in aqueous solution undergoes a swelling-to-shrinking phase transition because of breaking of the intermolecular hydrogen bonding between PNIPAM chains and water. Thus, PNIPAM networks swell greatly below the LCST but shrink above the

∗ Corresponding author. Tel.: +82 42 821 6615; fax: +82 42 821 8870. E-mail addresses: [email protected], [email protected] (T.S. Lee).

LCST, because of the reversible association/dissociation of water molecules with/from the polymer chains, respectively [13–18]. In addition, PNIPAM microgels with fluorescence-sensing ability have attracted much interest in various fields because these systems have many advantages, including high sensitivity, fast response, real-time monitoring, and operational simplicity [19–21]. Fluorescein and rhodamine fluorophores have been widely used to label and track cells, proteins, and organelles in the human body because of their excellent biocompatibility and high sensitivity under physiological conditions [22–24]. Furthermore, fluorescein and rhodamine derivatives are known to be ideal molecules for constructing fluorescent chemosensors because of their excellent photophysical properties, such as high absorption coefficients and high quantum efficiencies. Under basic or neutral pH conditions, fluorescein gives a strong green fluorescence with opening of its lactone ring, but the emission is remarkably reduced at acidic pH, induced by ring closure. In contrast, rhodamine shows strong orange fluorescence at acidic pH because of opening of its lactam ring but becomes non-fluorescent at basic or neutral pH when the ring is closed. Although fluorescein and rhodamine derivatives have been shown to display high fluorescence in each pH environment, their applications in ratiometric pH sensors have not been greatly explored. Recently, nanoparticle or polymer-based sensors conjugated with pH-sensitive fluorescein and rhodamine dyes were reported for detecting a wide range of pH values [25–28]. In this work, we have synthesized a microgel of poly(Nisopropylacrylamide-co-fluorescein-co-rhodamine) crosslinked with N,N -methylenebisacrylamide (PNFR microgel) via free

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radical emulsion co-polymerization. A wide range of pH could be monitored using the PNFR microgel, in which the fluorescein and rhodamine in the microgel showed efficient and independent emission changes over a wide range of pH. The fluorescein and rhodamine derivatives in the PNFR microgel gave green and orange fluorescence emissions at neutral to basic pH and at acidic pH, respectively. The changes in turbidity at temperatures above the LCST of the microgel enabled us to provide a possible monitoring of the temperature as well. The PNFR microgel exhibited a pHsensitive fluorescence change that was easy to observe with the naked eye. Moreover, because of the crosslinking, the microgel was stable enough to withstand repeated and reversible changes of the pH. To our knowledge, crosslinked PNIPAM copolymer containing two fluorophores has been rarely reported. Thus, this system envisaged possible monitoring of pH or temperature with a single polymeric material.

2. Experimental 2.1. Reagents and characterization All chemicals were purchased from Sigma–Aldrich and used without further purification. Methacryloyl chloride was purchased from Sigma–Aldrich and purified by vacuum distillation before use. 1 H NMR spectra were obtained using a Bruker DRX-300 spectrometer (Korea Basic Science Institute). The elemental analysis was performed using a CE Instruments EA-1110 elemental analyzer.

FT-IR analysis was carried out using a Bruker Tensor 27 spectrometer. The particle images were obtained using a field emission-scanning electron microscope (FE-SEM, Hitachi S-4800), and the size of the microgel was determined using dynamic light scattering (DLS, Malvern Zetasizer). UV–Vis absorption spectra were recorded on a PerkinElmer Lambda 35 spectrometer. Photoluminescence spectra were obtained with a Varian Cary Eclipse equipped with a xenon lamp excitation source.

2.2. Synthesis of N-(rhodamine B)lactam-ethylenediamine (1) N-(Rhodamine B)lactam ethylenediamine was synthesized according to the previous method [29]. In a 250 mL flask, rhodamine B (2.4 g, 5.42 mmol) was dissolved in 60 mL of ethanol, then ethylenediamine (6 mL, 89.85 mmol) was added dropwise with stirring at room temperature. After the addition, the reaction mixture was heated and refluxed until the color of the solution changed from red to orange. After 12 h, the reaction mixture solvents were removed under vacuum. 1 M HCl (100 mL) was added to the residue, and the pH of the mixture was adjusted to basic pH by addition of 1 M sodium hydroxide (150 mL). The resulting precipitate was filtered and washed three times with deionized water. After removing the solvent under vacuum, the crude product was recrystallized from acetonitrile/water to afford a whitish-pink solid (2.47 g, 94%). 1 H NMR (300 MHz, CDCl3 ): ı = 7.93–7.90 (d, 1H), 7.47–7.44 (m, 2H), 6.46–6.27 (m, 6H), 3.38–3.18 (m, 10H), 2.44–2.39

Scheme 1. Synthetic routes to monomer 2 and polymer.

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Fig. 1. SEM image of the PNFR microgel.

(t, 2H), 1.20–1.15 ppm (t, 12H). Anal. Calc. for C30 H36 N4 O2 : C, 74.3%; H, 7.49%; N, 11.5%, Found: C, 74.2%; H, 7.51%; N, 11.6%. 2.3. Synthesis of N-(rhodamine B)lactam ethyl-2-methacrylamide (2) N-(Rhodamine B)lactam ethyl-2-methacrylamide was synthesized as described in a previous report [21]. In a 100 mL flask, 1 (1 g, 2.06 mmol) and triethylamine (0.43 mL, 3.10 mmol) were dissolved in chloroform (40 mL), then the solution was cooled in an ice bath. A solution (5 mL) of methacryloyl chloride (0.3 mL, 3.10 mmol) in chloroform was added to the flask dropwise with stirring. After 18 h, the reaction mixture solvents were removed under vacuum, and then the crude product was dissolved in ethyl acetate. The organic mixture was extracted with NaHCO3 solution, brine, and water. The organic phase was dried over anhydrous magnesium sulfate, filtered and the solvent was removed under vacuum. The residue was purified by column chromatography to afford a pinkred solid (0.65 g, 57%). 1 H NMR (300 MHz, CDCl3 ): ı = 7.93–7.92 (d, 1H), 7.69–7.62 (t, 1H), 7.49–7.46 (m, 2H), 7.11–7.09 (d, 1H), 6.47–6.25 (m, 4H), 5.79 (s, 2H), 3.36–3.07 (m, 12H), 1.32 (s, 3H), 1.21–1.14 ppm (t, 12H), Anal. Calc. for C34 H40 N4 O3 : C, 73.8%; H, 7.29%; N, 10.14%, Found: C, 73.0%; H, 7.38%; N, 10.0%. 2.4. Synthesis of the PNFR microgel N-isopropylacrylamide (2.25 g, 19.88 mmol), N,N methylenebisacrylamide (0.03 g, 0.19 mmol), fluorescein O-methacrylate (0.01 g, 0.025 mmol), 2 (0.138 g, 0.25 mmol), and sodium dodecyl sulfate (0.73 g, 2.53 mmol) were dissolved in dry DMF (35 mL) and deionized water (55 mL) in a 250 mL flask under argon. A solution (10 mL) of ammonium persulfate (0.64 g, 2.8 mmol) in deionized water was added to initiate the

Fig. 2. Size distributions of PNFR microgel at (a) 25 ◦ C and (b) 40 ◦ C. (c) Changes in the diameters of pure PNIPAM microgel and the PNFR microgel according to the temperatures (determined by DLS).

emulsion polymerization and the reaction was carried out at 70 ◦ C for 4 h. After the reaction, the reaction mixture was cooled to room temperature and poured into deionized water (600 mL). The microgel was isolated by centrifugation (14,000 rpm) and washed three times with deionized water. Finally, the PNFR microgel was

Scheme 2. Structure transitions of fluorescein and rhodamine derivatives as a function of pH.

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Fig. 3. (a) Absorption and (b) emission spectra of the PNFR microgel (16.4 mg) at various pHs and temperatures in deionized water (10 mL). Arrow direction (green emission): pH 6.3, 7.2, 7.8, 8.4, 9.1, 10.3, and 11.6; (red emission): pH 2.7, 3.4, 3.9, 4.6, and 5.3. Absorption spectra were obtained at 25 ◦ C. Emission spectra were obtained using excitation wavelengths at 490 nm (for green emission) and 560 nm (for red emission). (For interpretation of the references to color in this legend, the reader is referred to the web version of the article.)

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Fig. 4. Fluorescence response of the PNFR microgel to temperature and pH in the (a) basic range (pH 9–11), (b) neutral range (pH 7) and (c) acidic range (pH 3–5). I514 and I586 correspond to the fluorescence intensity at 514 nm and 586 nm, respectively.

obtained by centrifugation to yield 0.82 g. FT-IR(cm−1 ) : 3437(O H), 3300(N H), 3076(sp2 C H), 2972(sp3 C H), 1649(C O), 1367(C N), 1128(C O).

3. Results and discussion The synthetic routes to the rhodamine-containing monomers and polymer are illustrated in Scheme 1. The PNFR microgel was obtained using free radical emulsion copolymerization and the crosslinking was carried out using N,N -methylenebisacrylamide. The chemical structure of the PNFR microgel was confirmed using FT-IR because of the crosslinked structure. The PNFR microgel was obtained as spherical nanoparticles with relatively narrow size distribution in aqueous solution, according to the SEM image of Fig. 1. Moreover the microgel exhibited a uniform hydrodynamic radius of 847 nm (PDI 0.014) at 25 ◦ C and 173 nm (PDI 0.005) at 40 ◦ C, which demonstrated a typical LCST behavior (Fig. 2a and b). The phase transition temperature of the newly synthesized PNFR microparticles was investigated using DLS as shown in Fig. 2c, which shows the phase transition temperature at 35.5 ◦ C, which seems to be very similar to that of pure PNIPAM because of small composition of the fluorophores. It is expected that fluorescence changes in the PNFR microgel would be caused by structural changes of fluorescein and rhodamine derivatives under a variety of pH as shown in Scheme 2. At basic or neutral pH conditions, the lactone ring of the fluorescein moiety is opened, and it gives a strong green fluorescence. At the same time, the lactam ring of the rhodamine moiety maintains its closed ring system with very weak emission. In contrast, at

acidic pH, the lactam ring of the rhodamine moiety is opened, and it gives a strong orange fluorescence. However, the lactone ring of the fluorescein moiety is closed, and thus it is non-fluorescent. The optical properties of the PNFR microgel in deionized water were investigated using UV–vis absorption and fluorescence spectroscopy at different pHs and temperatures (Fig. 3). Under basic and neutral pH conditions, the PNFR microgel showed an absorption band at 490 nm and strong fluorescence at 514 nm with green emission. In contrast, under acidic pH conditions, the PNFR microgel exhibited an absorption band at 560 nm and strong fluorescence at 586 nm, emitting orange light. The increase in pH gave rise to intensified fluorescence of the fluorescein moiety in the polymer at 514 nm, while the fluorescence intensity of the rhodamine moiety at 586 nm decreased gradually. Based on these results, the pHdependent fluorescence changes in the PNFR microgel could be used in monitoring the pH of a solution. To investigate the temperature dependence of the fluorescence changes on different pHs, the temperature of the microgel was varied ranging from 25 ◦ C to 42 ◦ C. It is known that the NIPAMcontaining PNFR microgel shows LCST behavior above 35.5 ◦ C, where the microgel solution becomes turbid near the LCST. Such changes in fluorescence above LCST are likely to result from the shrinkage of the gel structure, leading to the decrease in fluorescence intensity compared with that below LCST. Because of the increased turbidity in PNFR microgel over 35.5 ◦ C, a smaller increment (514 nm) or decrement (586 nm) can be observed over 35.5 ◦ C than the intensity values under 35.5 ◦ C in Fig. 3b. It is generally recognized that the ratiometric detection method is a reliable technique that can quantify the target analyte precisely [30,31]. In this case, ratiometric sensing of pH is possible because of the presence of two emission bands at 514 nm and 586 nm, resulting from fluorescein and rhodamine fluorophores,

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I514 / I586

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150 2

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pH Fig. 5. Changes in fluorescence ration (I514 /I586 ) of the PNFR microgel as a function of pH at 25 ◦ C. I514 and I586 correspond to the fluorescence intensity at 514 nm and 586 nm, respectively. Excitation wavelengths: 490 nm (for I514 ) and 560 nm (for I586 ).

respectively, in which the emission at 514 nm (fluorescein) is more affected at higher pH and the emission at 586 nm (rhodamine) is more influenced at lower pH. Therefore, the intensity ratio of the PNFR microgel at such wavelengths was employed as the sensing signal. Fig. 4 exhibits the fluorescence response to relationship between temperature and pH. In basic pH range, the fluorescence ratio (I514 nm /I586 nm ) tends to slightly decrease as increase in the temperature, while fluorescence ratio increases as the temperature increases in acidic pH. In this respect, the PNFR is responsive to dual change of temperature and pH. To elucidate further the pH-dependent emission change of the PNFR microgel, the relationship between emission intensity ratios and pH was investigated, as shown in Fig. 5. The microgel showed a linear relationship between the fluorescence ratio and pH in two pH windows such as 2–6 and 7–12. The photographic images in Fig. 6 show that use of the PNFR microgel is an easy and effective strategy for monitoring pH, by observing the fluorescent color change from orange to green upon increasing pH. Though the specific pH can be

Relative Intensity (514 nm)

0.015

Fig. 7. Fluorescence emission response of the PNFR microgel to pH cycles (pH 4 and 10). Inset photographs were taken under UV illumination at 365 nm. I514 and I586 correspond to the emission intensities at 514 nm and 586 nm, respectively.

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Fig. 8. Effect of the ionic strength on the fluorescence change of PNFR microgel in the presence of NaCl, KCl, CaCl2 (10 mM each). Relative fluorescence intensity (a) at 514 nm (pH 12) and (b) at 586 nm (pH 4). Excitation wavelengths for (a): 490 nm; for (b): 560 nm.

Fig. 6. Photographs of the PNFR microgels (16.4 mg) in 10 mL water at various pH under hand-held UV lamp (365 nm).

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recognized using spectrometer, pH windows (acidic or basic condition) can be regarded by the naked eye. The large pH-dependent changes in fluorescence color lead to the possible naked-eye monitoring of the pH of the aqueous solution. In addition, all signal changes are completely reversible as shown in Fig. 7. The reversible cycles of emission changes of the PNFR microgel between pH 4 and 12 could be repeated for 20 cycles with a negligible fluorescence bleaching, indicating that the microgel was stable under reversible changes in pH, mainly because of its crosslinked structure. Moreover, it is clearly indicated that the PNFR microgel was stable and maintained its initial fluorescence under different ionic strengths as shown in Fig. 8. The presence of electrolytes with various ionic strengths did not affect the fluorescence of the PNFR microgel, indicating that the fluorescein and rhodamine B moieties in the microgel were stable in the presence of electrolytes. To elucidate the effect of ionic strength of electrolytes on the fluorescence changes of PNFR, three kinds of electrolytes (sodium chloride, potassium chloride, and calcium chloride) were prepared at the concentration of 10 mM. The ionic strengths of such electrolytes were evaluated to be 10 mM (for sodium chloride and potassium chloride) and 30 mM (for calcium chloride). Fig. 8 shows that the kinds and ionic strength of electrolytes added did not affect the fluorescence of the PNFR microgel. Such results described above reveal that the designed microgel is suitable for the monitoring of pH or temperature with high performance. 4. Conclusion Thermo-responsive PNIPAM-based microgel labeled with a reversible fluorescent pH probe composed of both fluorescein and rhodamine derivatives was synthesized by free radical emulsion copolymerization. We confirmed that the lactone ring of fluorescein and the lactam ring of rhodamine open in neutral-basic and acidic conditions to provide fluorescence emissions at 514 nm and 586 nm, respectively. Furthermore, the PNFR microgel showed changes in fluorescence intensity because of the turbidity of the solution as the temperature changed. We also confirmed that the microgel showed a linear relationship between fluorescence change and pH in the pH ranges of both acidic and basic conditions such as 2.3–5.3 and 7.4–12.4. These fluorescence changes could be easily observed by the naked eye. Thus, the crosslinked PNFR microgel can be a potential candidate for sensitive fluorometric monitoring of pH or temperature in aqueous solution, which has many advantages for practical applications in sensing, imaging, and diagnostic systems. Acknowledgment Financial support from the National Research Foundation (NRF) of Korean government through Basic Science Research Program (2012R1A2A2A01004979) is gratefully acknowledged. References [1] Y. Zhang, A.L. Yarin, Stimuli-responsive copolymers of n-isopropyl acrylamide with enhanced longevity in water for micro- and nanofluidics, drug delivery and non-woven applications, J. Mater. Chem. 19 (2009) 4732–4739. [2] L. Peng, M. You, Q. Yuan, C. Wu, D. Han, Y. Chen, Z. Zhong, J. Xue, W. Tan, Macroscopic volume change of dynamic hydrogels induced by reversible DNA hybridization, J. Am. Chem. Soc. 134 (2012) 12302–12307. [3] N. Orakdogen, Design and synthesis of dual-responsive hydrogels based on N,N-dimethylaminoethyl methacrylate by copolymerization with Nisopropylacrylamide, Macromol. Res. 22 (2014) 32–41. [4] Z. Meng, G.R. Hendrickson, L.A. Lyon, Simultaneous orthogonal chemoligations on multiresponsive microgels, Macromolecules 42 (2009) 7664–7669. [5] Y. Guan, Y. Zhang, PNIPAM microgels for biomedical applications: from dispersed particles to 3D assemblies, Soft Matter 7 (2011) 6375–6384.

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Biographies Yongkyun Kim obtained his BSc in advanced organic materials and textile system engineering from Chungnam National University in 2013. Now he is studying in

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Organic and Optoelectronic Materials Laboratory at Chungnam National University. His current research interests are biomolecule recognition and nanohybrid sensor. Daigeun Kim obtained his master degree in department of nanotechnology from Chungnam National University in 2012. Now he is a PhD student at advanced organic materials and textile system engineering at Chungnam National University. His current research interests are nano-hybrid as sensory and biological materials. Geunseok Jang received his master degree from Chungnam National University in 2012. Now he is studying as a PhD student in organic and optoelectronic materials lab at Chungnam National University. His main fields are sensors and organic chemistry.

Jongho Kim obtained master degree in graduate school of analytical science and technology from Chungnam National University in 2013. Now he is studying in advanced organic materials and textile system engineering at Chungnam National University (PhD course). His current research interests are light emitting materials science and sensors chemistry. Taek Seung Lee received a BS in textile engineering from Seoul National University in 1988 where he obtained a PhD in fiber and polymer science in 1994. After postdoctoral researches at Korea Institute of Science and Technology and University of Massachusetts Lowell, he joined Chungnam National University as an assistant professor in 1997 and became a full professor in 2008. His research interest includes synthesis of functional organic materials and construction of hybrid nanomaterials, which have potential uses in optoelectronic materials and chemical/biological sensors.

Please cite this article in press as: Y. Kim, et al., Fluorescent, stimuli-responsive, crosslinked PNIPAM-based microgel, Sens. Actuators B: Chem. (2014), http://dx.doi.org/10.1016/j.snb.2014.10.089