Thermosensitive Behavior of Poly(N-isopropylacrylamide) and

The redox current of Hb gradually lowered when the prepared electrode was immersed into pH 7.0 PBS at 20 °C, indicating that incorporated Hb could be...
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Thermosensitive Behavior of Poly(N-isopropylacrylamide) and Release of Incorporated Hemoglobin Zhengzhi Yin, Jingjing Zhang, Li-Ping Jiang,* and Jun-Jie Zhu* Key Laboratory of Analytical Chemistry for Life Science (MOE), School of Chemistry and Chemical Engineering, Nanjing UniVersity, Nanjing 210093 (P. R. China) ReceiVed: April 19, 2009; ReVised Manuscript ReceiVed: July 21, 2009

A poly(N-isopropylacrylamide) (PNIPAm) brush has been successfully fabricated on an indium tin oxide (ITO) film via a simple electrochemical route. The polymer thermoresponsive behavior was investigated with an electrically heated ITO electrode. This kind of electrode demonstrated a rapid response to heating up and down, and the results indicated that the polymer modified interface possessed a characteristic lower critical solution temperature (LCST) and showed ON/OFF switch behavior. Furthermore, the model protein of hemoglobin (Hb) was incorporated into the polymer by a thermal “breathing-in” process. The electrochemical experiments revealed that the film could provide a friendly microenvironment for Hb to promote direct electron transfer. A pair of well-defined redox peaks with a formal potential of -204 mV (verses saturated calomel electrode, SCE) was observed. The redox current of Hb gradually lowered when the prepared electrode was immersed into pH 7.0 PBS at 20 °C, indicating that incorporated Hb could be released from the PNIPAm film, which proved that the approach could provide a potential route in the design of responsive biocompatible surfaces. 1. Introduction To date, increasing attention has been placed on the development of controlled switchable surfaces, also known as “smart surfaces”,1 which switch their physicochemical properties in response to external stimuli. Such reversibly switchable surfaces might be applied in controllable release matrices,2 permeation membranes,3 intelligent microfluidic switching and functional textiles,4 artificial muscles,5 biosensors,6 information storage,7 bioresponsive optical elements,8 temperature-induced switching,9 thermally responsive filters,4 and so on. Among these smart materials, temperature responsive poly(N-isopropylacrylamide) (PNIPAm) is one of the most important and intensively studied polymers.7,10,11 Interest in the PNIPAm system stems from the fact that it exhibits a fully reversible lower critical solution temperature (LCST) of 32 °C in aqueous solution, which can be attributed to the alterations in the hydrogen-bonding interactions of the amide group,4,12 and the polymer is attractive in biological applications because the temperature lies between room temperature and body temperature. Above the LCST, the polymer is collapsed and hydrophobic, whereas it is expanded and hydrophilic below the LCST. Furthermore, on the basis of the thermosensitive and biocompatible properties, intelligent PNIPAm can be used as an ideal material to bridge the gap between biological machines and multifunctional actuators.13 Some reports have involved grafting PNIPAm on solid surfaces and corresponding thermoresponsive properties.4,11,14,15 However, it is still a challenge for the design of an efficient approach to control the fabrication and in situ investigation of the sensitive interfaces.15 Electrically heated electrodes (HE) have attracted considerable attentionsincetheywereintroducedbyGru¨ndlerandco-workers.16-18 HE can be used to control the electrode temperature easily and * To whom correspondence should be addressed. E-mail: [email protected] (J.-J.Z.), [email protected] (L.-P.J.). Telephone and Fax: +86-25-83594976.

also stay the temperature of the bulk solution. This technique is based on symmetric electrode arrangement and special equipment for applying an alternate heating current with high frequency, which can avoid disturbances from the heating current. Recently, Chen and co-workers successfully designed an ECL detection system using a xanthine oxidase-modified electrically heated carbon paste electrode to determine hypoxanthine. The results showed that the detection limit at 35 °C was 30-fold lower than that at 25 °C.18 When the properties of the thermosensitive material were studied using the usual electrodes, we found one problem might be control of electrode temperature. With the technique of HE, this problem is easily solved. To the best of our knowledge, the investigation of the properties of the temperature responsive polymer-modified heated electrodes has not been reported. As one of the most important transparent conducting oxides, tin-doped indium oxide (ITO) has been found to have wide applications in photovoltaics, smart windows, organic light-emitting diodes, flat panel displays,19 and optic waveguide systems.20 In the field of electrochemistry, ITO films can usually be used as working electrodes. Because of its good transparency and low resistivity, it can be used in simultaneous optical investigations. Furthermore, thin ITO films have been used as transparent heaters in various applications such as outdoor panel displays, avionic displays, liquid crystal display panels for use in harsh environments, periscopes, and vehicle window defrosters.21 Then, it is significant to combine the advantages of ITO and the features of HE. It is very important for polymers to absorb and release biomolecules, especially in drug delivery. Lowman immobilized insulin in microgels of cross-linked poly(methacrylic acid) grafted with poly-(ethylene oxide) (PEO), which enhanced the hypoglycemic effects upon oral administration.22 Kabanov developed nanogels from cross-linked polyethyleneimine and PEO, which could deliver immobilized nucleotides across cellular barriers and protect them from degradation by metabolic systems within cells.23 Recent reference also reported the

10.1021/jp903589a CCC: $40.75  2009 American Chemical Society Published on Web 08/12/2009

Thermosensitive Behavior of Poly(N-isopropylacrylamide) exploration of polyelectrolyte network materials containing cross-linked poly(acrylic acid) and PEO chains for the adsorption and release of the model protein cytochrome C.24 Herein, an ITO HE has been successfully fabricated to study the thermoresponsive properties of the PNIPAm brush layer synthesized with electrochemically induced free-radical polymerization (EIFRP). Furthermore, hemoglobin (Hb) was chosen as a model protein to study the “breathing-in” and release properties of the polymer. The UV-vis spectra and electrochemical results indicated that the incorporated Hb retained its native structure, showing good direct electron transfer and electrocatalytic ability to hydrogen peroxide (H2O2).

J. Phys. Chem. C, Vol. 113, No. 36, 2009 16105 SCHEME 1: Scheme of ITO HE. (A) Glass, (B) ITO Film, (C) Insulating Tape, (D) Connection to AC Heating Device, and (E) Working Electrode Contact

2. Experimental Section Materials. N-isopropylacrylamide (NIPAm) (Aldrich) was recrystallized in hexane before it was used. Sodium nitrate (NaNO3), sodium persulfate (Na2S2O8) (Tianjin VAS Chemical Reagent Co.), and bovine hemoglobin (Hb) (MW 64500) (Sigma) were used as received without further purification. Hydrogen peroxide (H2O2) (30% w/v solution) was purchased from the Beijing Chemical Engineering Plant. Glass plates for the ITO film coating were purchased from Conduc Optics & Electronics Technology Co., Ltd. (sheet resistance ca. 7 Ω/0). Double-distilled water was used throughout all experiments. All other chemicals were of analytical grade and used without further purification. Apparatus. All electrochemical experiments were performed on a CHI 660 electrochemical analyzer (Co. CHI, U.S.A.) with a conventional three-electrode system comprised of platinum wire as the auxiliary electrode, saturated calomel electrode (SCE) as the reference, and prepared ITO as the working electrode. The buffer solution was purged with highly purified nitrogen for at least 30 min, and a nitrogen atmosphere environment was maintained in all electrochemical measurements. The heating current (100 kHz ac) was provided by a function generator (DF1027B, Ningbo Zhongce Electronics Co., Ltd., China) with 40 W peak power. The output was connected to the electrode via a transformer. The heating current was monitored by measuring the voltage drop of 1 Ω resistance serially connected to the electrode with an alternating current (ac) millivolt meter (DF1933, Ningbo Zhongce Electronics Co., Ltd., China). UV-vis experiments were performed with a Ruili 1200 photospectrometer (Peking Analytical Instrument Co., Peking, China). The attenuated total reflection Fourier transform infrared spectroscopic (ATR-FTIR) measurement was performed on a Bruker model VECTOR22 instrument. Atomic force microscopy (AFM) was operated in tapping mode in air at room temperature. Images were acquired at a scan rate of 1.5 Hz and tip force constant of 40 N/m. Construction of Electrically Heated Electrodes (HE). The structure of a HE is sketched in Scheme 1. One piece of glass (about 17 mm × 47 mm) with single-faced ITO was masked and etched (solution of Vhydrochloric acid:Vnitric acid:VH2O ) 5:1:1 for 1 h) to leave three joint conducting strips. The three ITO strips served as the electrical contacts. The central contact (Scheme 1E) provides the connection to the electrochemical workstation, with the other two (Scheme 1D) connected to the heating device (possess about 120 Ω resistance). The electrode area was controlled by insulating tape (Scheme 1C) covering the edge of ITO layers and was determined to be about 26 mm2. Prior to each set of measurements, the ITO surface was carefully washed in acetone, ethanol, and pure water with sonication and drying with nitrogen gas, and the fabricated ITO HE can be used repeatedly after ultrasonic washing.

Figure 1. Cyclic voltammograms of ITO electrode in the solutions: (a) 0.15 M NaNO3, (b) 0.15 M NaNO3 + 10 mM Na2S2O8, and (c) 0.15 M NaNO3 + 10 mM Na2S2O8 + 0.8 M NIPAm. Insert is curve a.

Preparation of Polymer-Modified Electrodes. In a typical process, in situ electrochemistry-induced free-radical polymerization was performed in a nitrogen saturated aqueous solution, which contained a monomer (NIPAm, 0.8 M), initiator (Na2S2O8, 10 mM), and NaNO3 (0.15 M).15 Polymerization can be initiated by applying a voltage of -700 mV or a cycled (100 mV s-1) potential between 100 and -800 mV. After polymerization, the electrodes were washed with double-distilled water to remove the nonreactive monomer. The resulting PNIPAm was attached to the ITO surface. 3. Results and Discussion Polymerization and Characterization. Cyclic voltammograms (CVs) of the ITO electrode in the different solutions are shown in Figure 1. In the solution of sodium nitrate, no voltammetric response was observed (Figure 1a). However, an obvious cathodic peak could be observed at -700 mV in the presence of sodium persulfate, and the peak current decreased after the addition of NIPAm (Figure 1b,c), which suggested that the reduction peak came from the reduction of the Na2S2O8 initiator. The current of the reduction peak greatly decreased with polymerization, indicating that the resultant polymer attached to the surface of the electrode and impeded the diffusion of the initiator. Scheme 2 depicts polymerization of N-isopropylacrylamide on the ITO electrode.14,15 Ambient S2O82- near the electrode surface was reduced to SO42-. Synchronously, a small part of

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Figure 2. Topographical AFM images of a PNIPAm-modified ITO surface in dry air.

SCHEME 2: Electrochemical Synthesis of PNIPAm at an ITO Electrode

the radical of the strong oxidant SO4-• could effectively escape.25 Then, the initiating sulfate radical anions of SO4-• reacted with the NIPAm monomer to produce the radicals of the monomer. The radical monomer induced polymerization. The polymer-modified surface could be fabricated by applying a voltage of -700 mV for 10 min or the voltammetric scan with the potential between 100 and -800 mV for 30 cycles. However, when the potential was more positive than the reduction potential of S2O82-, formation of a polymer is difficult, which suggests that the reduction of initiator ions (S2O82-) plays an important role in polymerization. Figure 2 displays the typical AFM images of the PNIPAmgrafted surface. It is found that the polymer chains have irregular pores. AFM images provided tridimensional morphology, and the presence of a scraggy surface confirmed the depth of about 10 µm. ATR-FTIR also confirms the formation of PNIPAm as shown in Figure S1 of the Supporting Information. For PNIPAm, the N-H stretch at about 3275 cm-1 and the CdO stretch at about 1630 cm-1 indicate the presence of amide groups in the polymer layer, and the characteristic doublet at 1385 and 1360 cm-1 suggests the presence of the isopropyl group.14,15 Thermosensitive Behavior of a PNIPAm Interface. Electrically heated electrodes have attracted great interest from theoretical and practical views.18 To expediently investigate the thermoresponsive properties of the polymer, the we fabricated HE as shown in Scheme 1. The corresponding temperature of the HE was measured according to the reports,18,26,27 on the basis of the well-known temperature coefficient of the ferro/ferricyanide couple. The standard potential of this couple shifted about 1.56 mV K-1 in the negative direction. Measurements of the open circuit potential of this reversible redox couple during heating resulted in a linear relation between the square of the heating current and temperature, which indicated that temperature could be adjusted arbitrarily in a reproducible manner. A good linearity was obtained with a regression coefficient of about

Figure 3. Dependence of the temperature increase at the ITO HE on the square of heating currents.

0.9965 in the range between 0 and 35 °C as shown in Figure 3. Figure S2 of the Supporting Information displays the measurement of temperature increase by the open circuit method at an ITO HE versus time profile during heating. After heating, the electrode temperature increased from the bulk temperature to a steady value within the following about 60 s. Thermal stability was tested by heating the electrodes consecutively for a total of 10 min at two different temperatures. After reaching a steady value, the temperature fluctuation was only about 1 °C, showing a stable thermal response on the electrode for a relatively long heating time. Cyclic voltammograms of Fe(CN)63-/4- at the bare ITO HE with various temperatures are shown in Figure 4. The peak potential separation (∆Ep) is 100 mV, and the formal potential E° is 224 mV for this redox couple at room temperature. The peak current is proportional to the square root of the potential sweep rate, which shows that the electrochemical process is controlled by diffusion. The peak current increased, and the redox potential shifted to the negative, along with the elevated temperature as shown in Figure 4 (insert A). When the electrode

Thermosensitive Behavior of Poly(N-isopropylacrylamide)

Figure 4. Cyclic voltammograms of the bare ITO HE in 0.5 M KCl aqueous solution containing 5.0 mM Fe(CN)63-/4- at different temperatures: (a) 15, (b) 26.8, (c) 33.2, (d) 39.6, (e) 49.3, and (f) 55.7 °C. Scan rate: 100 mV s-1. Insert A: cathodal peak current (black) and anodic peak current (red) versus electrode temperature. Insert B: anodic potential (red), formal potential (green), and cathodal potential (black) versus electrode temperature.

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Figure 6. Thermal response reduction peak currents of Fe(CN)63-/4at a PNIPAm-functionalized interface at variable temperatures.

Figure 7. Reduction peak current changes of Fe(CN)63-/4- upon switching the temperatures of a PNIPAm-modified electrode.

Figure 5. Cyclic voltammograms of the PNIPAm-functionalized ITO HE at variable temperatures. (a-j) 15, 21, 25.5, 27.3, 28.5, 31, 33.9, 37.1, 42, and 45 °C. Data recorded in 5.0 mM Fe(CN)63-/4- plus 0.5 M KCl aqueous solution. Scan rate: 100 mV s-1.

SCHEME 3: Schematic of Temperature-Induced Switchable PNIPAm Brush-Modified Interfaces

was heated, the temperature of the electrode surface increased rapidly, which might be attributed to the thicker layer of temperature distribution on the ITO surface than on the diffusion layer. Therefore, a relative high temperature on the electrode surface not only speeds up the diffusion but also induces convection. As a result, mass transfer is greatly improved on the electrode surface, and a larger current can be observed at the bare ITO electrode. The linear dependence plot for the peak potential and formal potential versus temperature is in accordance with the theoretical value of the electrode potential dependence of the temperature (Figure 4, insert B). Conformational changes play an important role in a wide variety of PNIPAm-based systems, and considerable efforts have been devoted to characterizing the changes of PNIPAm chains and volume changes of its gels.28 Here, the electrochemical technique was applied to investigate the conformational changes directly on the polymer surface as a function of temperature.

The representative CVs at various temperatures were recorded on the PNIPAm-modified electrode in 5 mM Fe(CN)63-/4- as shown in Figure 5. Redox currents increased from 15 to 21 °C, indicating that the diffusion and convection effect played a dominant role. However, the currents decreased from 21 to 45 °C due to the collapse of the polymer chains on the ITO surface, which showed that the changes caused by the increase in the electrode temperature was unfavorable for electron transfer between the redox label and electrode surface as shown in Scheme 3. A corresponding plot of reduction currents of Fe(CN)63-/4on the polymer-modified interface versus temperature is shown in Figure 6. Clearly, the phase transition temperature (LCST) of the attached polymer was estimated to be about 31 ( 2 °C, which further verified that the polymer has been attached on the ITO. The results meant that the responsive polymer interfaces could convert the environmental information effectively into an electrochemical signal, which might have potential applications in bioelectronics.29,30 When the temperature was repeatedly switched between 20 and 40 °C, the reduction currents of Fe(CN)63-/4- at the polymermodified electrode can be repeatedly cycled, showing that the states with different interfacial properties were switchable as shown in Figure 7. The resulting ON and OFF cycles of currents can be interpreted as the swelling and shrinking changes of PNIPAm below and above its LCST. This property of the polymer may have potential applications in protein separation, layer-by-layer assembly, and controlled release. The switching performance also exhibited good reversibility and quick transformation as a single cycle measurement for only a few minutes. This reversibility still remained after the polymer modified ITO HE was dipped in water without special protection for months, which indicated that the polymer film was stable. However, an ultrasonic treatment could cause rapid detachment of the

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Figure 8. UV-vis spectra of Hb incorporated into the PNIPAm attached on ITO, showing disposal (a) without Hb, (b) at 40 °C, and (c) with 20 °C water.

polymer films from the ITO surface. Interestingly, the polymer was translucent when the electrode temperature was below the LCST and was blurry above the LCST, which could be used in controlling the transparency of the materials. Application of the Phase Transition Property of the Polymer. The resulting ON and OFF phenomena have potential applications in controlled ion and molecule permeation and bioelectronics.30,31 The synthesized polymer was allowed to swell 3 mg mL-1 hemoglobin in solution at 20 °C. After the swelling, the slide was taken out of the Hb solution, immersed in pure water of 40 °C by shrinking to wash off the external protein, and then dried at 40 °C. The positions of the Soret absorption band of heme may provide information about the possible denaturation of the heme protein and particularly the conformational change in the heme group region.32 Figure 8 showed UV-vis absorbance spectra of above polymer-grafted ITO. The characteristic Soret absorption band of Hb at 408 nm indicated that Hb retained its native conformation in the polymer. The resulting polymer was immersed in pure water at 20 °C to swell, and then the absorbance of the Soret band decreased in intensity as shown in Figure 8c. Presumably, most of the incorporated Hb was physically inhaled into the brush film and could be released when the polymer presented an expanding state (Scheme 3). The residual absorbance at 408 nm suggested that a little Hb was entrapped by the polymer film. Remarkably, the permeability of Hb was significantly controlled by an ON/OFF manner. Controlled experiments revealed that the treatment of the shrunk polymer (40 °C) with the Hb solution did not lead to the incorporation of the molecules into the polymer. All of these results illustrated that the brush had the “breathing-in” and release property. The permeation of Hb into the polymer could be successfully controlled by phase transition of the polymer chains. In general, the potentials of redox proteins in thin films depend on the interactions of the proteins with film material and electrode material.33 When the resulting polymer films were immersed in a pH 7.0 buffer solution, a couple of well-defined and quasi-reversible reduction-oxidation peaks at -252 and -156 mV (vs SCE) were observed as shown in Figure 9. Obviously, the redox peaks coincided well with the potential characteristic of Fe(III)/Fe(II) redox couples of heme proteins. The cathodic and anodic peaks were nearly symmetric, and the reduction and oxidation peaks had roughly equal heights. This behavior suggested that all of the electroactive HbFe(III) within the films was converted to HbFe(II) on the forward scan to negative potentials, with full conversion of HbFe(II) to HbFe(III) on the reverse scan. The formal potential of the heme Fe(III/II) couple in the polymer-modified electrode was -204 mV in 0.1 M PBS (pH 7.0), which was similar to those reported previ-

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Figure 9. Cyclic voltammograms of the PNIPAm-modified ITO electrode incorpated with Hb. Data recorded in 0.1 M pH 7.0 PBS. Scan rate: 100 mV s-1.

Figure 10. Cyclic voltammograms of polymer/ITO electrode without (a) H2O2 and (b) 0.4 mM H2O2. Hb/polymer film without (c) H2O2 and (d) 0.2 mM H2O2. Data recorded in 0.1 M pH 7.0 PBS. Scan rate: 100 mV s-1.

ously.34 CVs showed that the reduction peak current of Hb decreased with an increase in cycle numbers, and the rates gradually slowed down. The phenomena were consistent with the release of Hb from the brush film and the growing of the commutative balance within the electrode surface as shown in Scheme 3. Electrocatalytic activity of the Hb/polymer-modified ITO electrode toward hydrogen peroxide was investigated. When H2O2 was added into pH 7.0 PBS, an increase in the reduction peak and decrease in the oxidation peak were observed. In the meantime, no reduction peak was observed in the presence of H2O2. With direct electrochemical reduction of Fe(III)Hb to Fe(II)Hb, Fe(II)Hb could be oxidized by H2O2 to Fe(III)Hb quickly, which could increase the reduction current to form a catalytic current. The typical catalytic reduction peak of the H2O2 was observed at -450 mV in Figure 10, and these results indicated that Hb preserved its catalytic activity. 4. Conclusions Thermosensitive behavior of poly(N-isopropylacrylamide) and release of incorporated hemoglobin were investigated. An electrically controlled heating ITO electrode was fabricated with rapid responses to heating up and down. Electrodes were used to investigate the thermal property of attached PNIPAm. The results revealed that the polymer interface exhibited ON/OFF switching behavior and a “breathing in” process, which indicated that the novel interfacial structures and thermoswitchable properties could be effectively controlled. Results suggested that polymer films provided a favorable microenvironment for Hb to transfer electrons directly. Most of the incorporated Hb was

Thermosensitive Behavior of Poly(N-isopropylacrylamide) physically inhaled into the brush film, which could be released when the polymer presented an expanding state. The integration of redox-active biomolecules into such a “smart” polymer could open the way to artificial models of biological redox systems leading to bioelectronic functionalities. The finding increases the understanding of thermosensitive polymers and might have a great potential in designing adaptive and responsive biocompatible surfaces in a variety of areas such as intelligent biological interfaces, controllable drug release, biosensors, membrane separation, temperature-controlled microfluidic switches, and so on. Acknowledgment. We greatly appreciate the support of the National Natural Science Foundation of China for Key Program (20635020), Creative Research Group (20821063), and General Program (20605011). This work is also supported by 973 Program (2006CB933201 and 2007CB936404). Supporting Information Available: Figure S1: In situ ATRFTIR of PNIPAm on ITO surface. Figure S2: Thermal stability of bare ITO HE during about a 10 min heating. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Milner, S. T. Science 1991, 251, 905–914. (2) Willner, I.; Rubin, S.; Shatzmiller, R.; Zor, T. J. Am. Chem. Soc. 1993, 115, 8690–8694. (3) Chu, L.; Li, Y.; Zhu, J.; Chen, W. Angew. Chem., Int. Ed. 2005, 44, 2124–2127. (4) Sun, T. L.; G., J.; Wang, Feng, L.; Liu, B. Q.; Ma, Y. M.; Jiang, L.; Zhu, D. B. Angew. Chem., Int. Ed. 2004, 43, 357–360. (5) Okuzaki, H.; Funasaka, K. Macromolecules 2000, 33, 8307–8311. (6) Lu, X. B.; Hu, J. Q.; Yao, X.; Wang, Z. P.; Li, J. H. Biomacromolecules 2006, 7, 975–980. (7) Luzinova, I.; Minkob, S.; Tsukruk, V. V. Prog. Polym. Sci. 2004, 29, 635–698. (8) (a) Kim, J.; Nayak, S.; Lyon, L. A. J. Am. Chem. Soc. 2005, 127, 9588–9592. (b) Kim, J.; Singh, N.; Lyon, L. A. Angew. Chem., Int. Ed. 2006, 45, 1446–1449. (9) Valkama, S.; Kosonen, H.; Ruokolainen, J.; Haatainen, T.; Torkkeli, M.; Serimaa, R.; Brinke, G. T.; Ikkala, O. Nat. Mater. 2004, 3, 872–876. (10) Lahann, J.; Mitragotri, S.; Tran, T.; Kaido, H.; Sundaram, J.; Choi, I. S.; Hoffer, S.; Somorjai, G. A.; Langer, R. Science 2003, 299, 371–374. (11) Sheeney-Hai-Ichia, L.; Sharabi, G.; Willner, I. AdV. Funct. Mater. 2002, 12, 27–32. (12) (a) Katsumoto, Y.; Tanaka, T.; Sato, H.; Ozaki, Y. J. Phys. Chem. A 2002, 106, 3429–3435. (b) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163–249. (13) Moya, S.; Azzaroni, O.; Farhan, T.; Osborne, V. L.; Huck, W. T. S. Angew. Chem., Int. Ed. 2005, 44, 4578–4581.

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