Azobenzene-Containing Molecularly Imprinted Polymer Microspheres

May 28, 2012 - ... Template Binding Properties in Pure Aqueous Media by Atom ..... addition fragmentation chain transfer strategy and its application ...
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Azobenzene-Containing Molecularly Imprinted Polymer Microspheres with Photo- and Thermoresponsive Template Binding Properties in Pure Aqueous Media by Atom Transfer Radical Polymerization Liangjing Fang, Sujing Chen, Xianzhi Guo, Ying Zhang, and Huiqi Zhang* Key Laboratory of Functional Polymer Materials, Ministry of Education, Department of Chemistry, Nankai University, Tianjin 300071, People’s Republic of China S Supporting Information *

ABSTRACT: A facile, general, and highly efficient approach to obtain azobenzene (azo)-containing molecularly imprinted polymer (MIP) microspheres with both photo- and thermoresponsive template binding properties in pure aqueous media is described for the first time, which involves the first synthesis of “living” azocontaining MIP microspheres with surface-immobilized alkyl halide groups via atom transfer radical precipitation polymerization (ATRPP) and their subsequent modification via surface-initiated atom transfer radical polymerization (ATRP) of N-isopropylacrylamide (NIPAAm). The successful grafting of poly(NIPAAm) (PNIPAAm) brushes onto the obtained MIP microspheres was confirmed by FT-IR, SEM, water dispersion stability and static contact angle studies, and template binding experiments. The introduction of PNIPAAm brushes onto the azo-containing MIP microspheres significantly improved their surface hydrophilicity and imparted thermoresponsive properties to them, leading to their pure water-compatible and thermoresponsive template binding properties. In addition, the binding affinity of the imprinted sites in the grafted azo-containing MIP microspheres was found to be photoresponsive toward the template in pure water, and this photoregulation process proved to be highly repeatable under photoswitching conditions.

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INTRODUCTION Molecular imprinting is a versatile and straightforward approach to generate artificial receptors with tailormade recognition sites for a given targeted molecule.1−6 It typically involves the copolymerization of a functional monomer and a cross-linker in the presence of a template molecule and a suitable porogenic solvent. The subsequent removal of the template molecule from the resulting cross-linked polymer networks leads to molecularly imprinted polymers (MIPs) with imprinted cavities complementary to the template in size, shape, and functionality, which can now specifically recognize and bind this target molecule. This, together with the favorable mechanical, thermal, and chemical stability of the MIPs, as well as their low cost, makes them very promising substitutes for biological receptors.1−6 Despite significant progress made in the molecular imprinting field in recent years, many challenges still remain to be addressed. It is known that the biological receptors such as enzyme and antibody can show outstanding molecular recognition abilities in aqueous environments and high responsivity toward external stimuli (e.g., temperature). The presently developed MIPs, however, are normally only organic solvent-compatible, and they mostly fail to show specific binding in pure aqueous media.7,8 Moreover, their high crosslinking characteristics (usually required to stabilize the binding © 2012 American Chemical Society

sites) make them normally difficult to deform for regulating their binding properties. These drawbacks significantly limit the potential of the MIPs in many applications such as biotechnology9 and intelligent drug delivery,10 because only those advanced functional MIPs with water-compatible and/or stimuli-responsive template binding properties can meet their requirements. To address these issues, much effort has been devoted to the development of water-compatible and stimuli-responsive MIPs. So far, some MIP hydrogels with stimuli-responsive template binding properties in aqueous media have been prepared by adding certain amounts of special responsive hydrophilic (co)monomers (e.g., N-isopropylacrylamide (NIPAAm), acrylic acid, or azo monomer) into molecular imprinting systems and carefully choosing the types and amounts of the cross-linkers used.11−15 In this context, it is worth mentioning that, among various stimuli-responsive MIPs (including both water-compatible and water-incompatible ones) developed up to now, photoresponsive ones have drawn particular attention because light stimulus can be imposed instantly and delivered in specific amounts with high accuracy.10 Although many previous reports Received: March 30, 2012 Revised: May 27, 2012 Published: May 28, 2012 9767

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the application of ATRP in the controlled synthesis of MIPs,26−33 to our knowledge, no water-compatible and stimuli-responsive MIPs have been prepared via this technique up to now. The chemical structures, morphologies, particle sizes, and surface hydrophilicity of the resulting MIP microspheres were characterized, and their water-compatible and dually stimuli-responsive template binding properties were investigated.

have demonstrated the great potential of photoresponsive azocontaining MIPs in smart separation and assays as well as controlled drug delivery, the MIPs with photoresponsive template binding properties in aqueous media are still scare.10 Lam and co-workers described the only report on the preparation of water-compatible photoresponsive MIP hydrogels for the photoregulated release and uptake of pharmaceuticals in the aqueous media.13 However, a water-soluble azo functional monomer had to be used in their work, which largely limits the general application of the method, because the most commonly used azo functional monomers are water-insoluble ones.16−21 In addition, the bulk hydrogel physical format of the resulting MIPs as well as the irregular shapes and relatively large sizes (normally tens of micrometers in diameter after the time-consuming and laborious grinding and sieving of the bulk MIP hydrogels) are inappropriate for such applications as smart binding assays and drug delivery, because the best physical format for such purposes is spherical beads.22,23 Furthermore, the binding sites inside the MIP particles with relatively large sizes are inaccessible, thereby significantly lowering the template loading capacities of the MIP particles. In particular, it is noteworthy here that it is still a challenging task to develop photoresponsive MIPs that can respond to other external stimuli in the meantime, although such advanced MIPs are highly desirable in many applications.24 Therefore, the development of versatile approaches to obtain micrometersized azo-containing MIP spherical particles with both photoresponsive and other stimuli-responsive template binding properties in aqueous solutions is of strong interest. In this paper, we describe for the first time a facile, general, and highly efficient approach to obtain azo-containing MIP microspheres with both photo- and thermoresponsive template binding properties in pure aqueous media, which involves the first synthesis of “living” photoresponsive azo-containing MIP microspheres with surface-immobilized alkyl halide groups via atom transfer radical precipitation polymerization (ATRPP) of an easily available pyridine group-containing azo functional monomer and their subsequent controlled surface-grafting of thermoresponsive poly(NIPAAm) (or PNIPAAm) brushes via surface-initiated atom transfer radical polymerization (ATRP) (Scheme 1). ATRP has proven highly versatile for the synthesis of “living” polymers with various well-defined structures.25 Although recent years have witnessed considerable interest in

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MATERIALS AND METHODS

Materials. Ethylene glycol dimethacrylate (EGDMA, Alfa Aesar, 98%) was purified by distillation under vacuum. Acetonitrile (Tianjin Jiangtian Chemicals, China, analytical grade (AR)) was refluxed over calcium hydride (CaH2) and then distilled. Tetrahydrofuran (THF, Tianjin Jiangtian Chemicals, 99%) was refluxed over sodium and then distilled. N,N-Dimethylformamide (DMF, Tianjin Jiangtian Chemicals, 99.5%) was dried with anhydrous magnesium sulfate (MgSO4) and then distilled under vacuum. Methanol (Tianjin Jiangtian Chemicals, AR) was distilled prior to use. NIPAAm (Acros, 99%) was recrystallized from hexane prior to use. Copper(I) chloride (CuCl, Tianjin Jiangtian Chemicals, AR) was purified by stirring it with acetic acid for 12 h, washed with ethanol and diethyl ether, and then dried under vacuum at 75 °C for 3 days. Tris(2-(dimethylamino)ethyl)amine (Me6TREN) was prepared by a one-step synthetic procedure from commercially available tris(2-aminoethyl)amine (TREN, Acros, 97%) according to the reported procedure.34 Methacrylate azo functional monomer 4-((4-methacryloyloxy)phenylazo)pyridine (MAzoPy) was prepared according to our previously reported procedure (Scheme S1 in the Supporting Information).21 2,4-Dichlorophenoxyacetic acid (2,4-D, Alfa Aesar, 98%), 2,4-dichlorophenylacetic acid (DPAc, Acros, 99%), phenoxyacetic acid (POAc, Acros, 98+%), 2,4dichlorophenol (2,4-DCP, Tianjin heowns Biochemical Technology Co., Ltd., China, 99%), 2,4-dichlorobenzaldehyde (2,4-DCAD, Tianjin heowns Biochemical Technology Co., Ltd., 98%), anhydrous copper(II) chloride (CuCl2, Alfa Aesar, 98%), ethyl 2-chloropropionate (Alfa Aesar, 97%), and all the other reagents were commercially available and used as received. The chemical structures of 2,4-D, DPAc, POAc, 2,4-DCP, and 2,4-DCAD are shown in SI Scheme S2. Synthesis of “Living” Azo-Containing MIP and NonImprinted or Control Polymer (CP) Microspheres with Surface-Immobilized ATRP Initiating Groups (i.e., Alkyl Halide Groups). The “living” azo-containing MIP microspheres with surfaceimmobilized alkyl halide groups were directly synthesized via ATRPP by using 2,4-D, MAzoPy, EGDMA, ethyl 2-chloropropionate, CuCl/ Me6TREN, and acetonitrile as the template, functional monomer, cross-linker, initiator, catalyst, and porogenic solvent, respectively, according to the following procedure: MAzoPy (0.4005 g, 1.5 mmol), 2,4-D (0.3316 g, 1.5 mmol), and dried acetonitrile (150 mL) were added into a one-neck round-bottom flask (250 mL) successively. A clear homogeneous solution was obtained after the reaction mixture was stirred at ambient temperature for 3 h in the dark. EGDMA (0.85 mL, 4.5 mmol) was then added into the reaction system. CuCl (5.19 mg, 0.0525 mmol) was added into the solution after the reaction mixture was purged with argon for 20 min. The reaction mixture was further purged with argon for 10 min, and then Me6TREN (0.036 g, 0.1575 mmol) was added. After another 10 min of argon bubbling, ethyl 2-chloropropionate (6.66 μL, 0.0525 mmol) was added into the system. The flask was then sealed and immersed into a thermostatted oil bath at 60 °C for 48 h in the dark. The resulting polymer particles were collected by centrifugation, which were purified through Soxhlet extraction with methanol/acetic acid (9/1 v/v, 48 h) and acetonitrile (48 h) successively to remove both the template and copper catalyst and then dried at 40 °C under vacuum overnight to provide the khakicolored azo-containing MIP particles (yield: 20%). The corresponding CP particles (khaki color) were prepared and purified under identical conditions except that the template was omitted (yield: 28%). Synthesis of Azo-Containing MIP/CP Microspheres with Surface-Grafted PNIPAAm Brushes. The azo-containing MIP/CP

Scheme 1. Schematic Protocol for the Synthesis of AzoContaining MIP Microspheres with Both Photo- and Thermoresponsive Template Binding Properties in Aqueous Media by ATRP

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microspheres with surface-grafted PNIPAAm brushes were synthesized via surface-initiated ATRP by using the above-obtained “living” azocontaining MIP/CP microspheres as the immobilized ATRP initiator according to the following procedure: The “living” azo-containing MIP/CP microspheres with surface-immobilized alkyl halide groups (100 mg), NIPAAm (0.99 g, 8.77 mmol), CuCl2 (2.01 mg, 0.015 mmol), and isopropanol (4 mL) were added into a one-neck roundbottom flask (25 mL) successively. CuCl (14.9 mg, 0.15 mmol) was then added into the system after the reaction mixture had been purged with argon for 20 min. The reaction mixture was further purged with argon for 10 min, and then Me6TREN (0.0377 g, 0.165 mmol) was added. After another 10 min of argon bubbling, ethyl 2chloropropionate (0.53 μL, 0.0043 mmol) was added into the system. The flask was then sealed and stirred at 25 °C for 24 h. After centrifugation, the resulting solid products were thoroughly washed with methanol until no white sediment was detectable when ether was added into the washing solutions, which were then dried at 30 °C under vacuum to the constant weights, leading to MIP and CP microspheres bearing polymer brushes with weight of 107 and 106 mg, respectively. The addition of some sacrificial initiator ethyl 2-chloropropionate into the above polymerization systems also led to the generation of free PNIPAAm in the reaction solutions, which were obtained by precipitating the supernatant solutions (they were obtained by the centrifugation of the polymerization solutions and were further purified by passing through a column of neutral aluminum oxide in order to remove the catalyst prior to the precipitation) into ether, filtered, and then dried at 30 °C under vacuum for 48 h. Characterization. Fourier transform infrared (FT-IR) spectra of the ungrafted and grafted azo-containing MIP/CP microspheres were obtained using a Nicolet Magna-560 FT-IR spectrometer. The morphologies, particle sizes, and size distributions of the ungrafted and grafted azo-containing MIP/CP microspheres were determined with a scanning electron microscope (SEM, Shimadzu SS550). The SEM size data reflect an average of all the particles in the SEM images, which are calculated by using the following formulas: k

Dn =

k

∑ niDi /∑ ni i=1

i=1

k

Dw =

Equilibrium binding experiments were performed by incubating a 2,4-D solution in acetonitrile (0.5 mL, 0.05 mM) or in pure water (0.5 mL, 0.05 mM) with different amounts of azo-containing MIP/CP microspheres at 25 °C for 6 h either in the dark or under the irradiation of 365 nm UV light (16 W). The amounts of the template bound to the MIP/CP microspheres were then quantified with HPLC (Scientific System Inc., USA) equipped with a UV−vis detector. The wavelength used for the determination of 2,4-D was 284 nm. A mixture of methanol and 0.5% aqueous solution of acetic acid (4/1 v/ v) was used as the mobile phase at a flow rate of 1 mL/min. Equilibrium binding experiments were also performed by incubating a 2,4-D solution in pure water (0.5 mL, 0.05 mM) with different amounts of the grafted MIP/CP microspheres at 45 °C for 6 h. Moreover, the equilibrium template bindings of the grafted MIP/CP microspheres (2 mg) were also determined by incubating them with a 2,4-D solution in pure water (0.5 mL, 0.05 mM) at different temperatures for 6 h in order to get more insight into the effect of the temperature on the binding properties of the grafted MIP/CP microspheres. The studies on the photoregulated release and uptake of the template 2,4-D and its structurally related analogues (i.e., DPAc and POAc) by the grafted azo-containing MIP microspheres were performed by alternately switching on and turning off the UV light irradiation on the mixtures of MIP microspheres and an aqueous solution of analytes: A series of samples were prepared by adding 2 mg of grafted azo-containing MIP microspheres and a mixed solution of 2,4-D, DPAc, and POAc in pure water (0.5 mL, C2,4‑D, DPAc or POAc = 0.05 mM) into plastic Eppendorf tubes (2 mL), respectively, which were subsequently sealed and put into an incubator equipped with a 365 nm UV lamp (16 W). After their incubation at 25 °C in the dark for 6 h, one sample was taken out from the incubator and centrifuged to determine the amounts of the analytes bound by the MIP microspheres (by HPLC). The UV light was then switched on in the incubator to irradiate the remaining samples immediately after the first sample was taken out. After 3 h of UV light irradiation with incubation for the samples at 25 °C, the UV light was switched off and the second sample was taken out for the determination of the bindings of the analytes. The remaining samples were then incubated at 25 °C in the dark for another 18 h, and the third sample was taken out for the determination of the bindings of the analytes. The UV light was then switched on again to irradiate the remaining samples immediately at 25 °C after the third sample was taken out. The above photoswitching procedures (i.e., UV light on for 3 h and off for 18 h alternately) were repeated until all of the other samples were measured. The studies on the photoregulated release and uptake of the template 2,4-D and its analogues DPAc and POAc by the azocontaining CP microspheres were carried out similarly. The binding selectivity of the grafted azo-containing MIP/CP microspheres was evaluated by measuring their competitive binding capacities toward 2,4-D and its two series of structurally related compounds in aqueous media: 2 mg of the grafted azo-containing MIP/CP microspheres were incubated with 0.5 mL of a mixed aqueous solution of 2,4-D, DPAc, and POAc (C2,4‑D, DPAc or POAc = 0.05 mM) or a mixed aqueous solution of 2,4-D, 2,4-DCP, and 2,4-DCAD (C2,4‑D, 2,4‑DCP or 2,4‑DCAD = 0.05 mM) at 25 °C for 6 h in the dark. The concentrations of the template 2,4-D and its related compounds DPAc and POAc were quantified with HPLC at a wavelength of 272 nm. A mixture of methanol and 0.5% aqueous solution of acetic acid (4/1 v/v) was used as the mobile phase at a flow rate of 1 mL min−1. On the other hand, the concentrations of 2,4-D, 2,4-DCP, and 2,4-DCAD were quantified with HPLC at a wavelength of 284 nm, and a mixture of methanol and 0.5% aqueous solution of acetic acid (6/4 v/v) was used as the mobile phase at a flow rate of 1 mL min−1. The “imprinting-induced promotion of binding” (IPB) was used to demonstrate the specificity of the studied MIP due to the molecular imprinting effect, which can be calculated by using the following equation:8,35

k

∑ niDi4 / ∑ niDi3 i=1

i=1

U = Dw /Dn where Dn is the number-average diameter, Dw the weight-average diameter, U the size distribution index, k the total number of the measured particles, Di the particle diameter of the ith polymer microsphere, and ni the particle number of the microspheres with a diameter Di. The molecular weights and polydispersity indices (PDIs) of the free polymers generated in the polymerization solutions during the surfaceinitiated ATRP processes (due to the addition of sacrificial ATRP initiator) were determined by using a gel permeation chromatograph (GPC) equipped with an Agilent 1200 series manual injector, an Agilent 1200 high-performance liquid chromatography (HPLC) pump, an Agilent 1200 refractive index detector, and three Waters UltraStyragel columns with 5−600K, 500−30K, and 100−10K molecular ranges (the temperature of the column oven was 35 °C). THF was used as the eluent at a flow rate of 1 mL/min, and the calibration curve was obtained by using polystyrene (PS) standards. The suspensions of the ungrafted and grafted MIP/CP microspheres in pure water (1 mg/mL) were first dispersed by ultrasonication, and they were then allowed to settle down for different times at 25 °C to check their dispersion stability. The polymer films of the ungrafted and grafted MIP/CP microspheres were prepared by casting their suspension solutions in DMF (10 mg/mL, after ultrasonic dispersion) on clean glass surfaces. After the solvent was allowed to evaporate at ambient temperature overnight, a KRÜ SS FM40 Easy Drop contact angle instrument (Germany) was utilized to determine their static water contact angles. Two measurements were taken across each sample, with their average being used for analysis.

IPB (%) = [(BMIP − BCP)/BCP] × 100% 9769

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where BMIP and BCP are the equilibrium bindings of the studied MIP and its corresponding CP toward an analyte, respectively. The larger the IPB value of the MIP toward the analyte, the better the selectivity of the MIP.

catalytic stability and activity in the presence of the acidic template. As a reference, the corresponding azo-containing nonimprinted or control polymer (CP) particles were also prepared similarly by omitting the template in the reaction system. All the reactions were performed at 60 °C in an argon atmosphere in the dark (in order to keep the azo moieties in their trans-rich state, Scheme 1), and the resulting polymer particles were thoroughly purified through Soxhlet extraction with methanol/acetic acid (9/1 v/v) and acetonitrile successively, leading to khaki-colored MIP and CP particles. The khaki color of the MIP and CP particles indicated that the azo functional moieties were successfully incorporated into the resulting polymers. The above-obtained azo-containing MIP/CP particles were first characterized with SEM (Figure 1a and b), and the results

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RESULTS AND DISCUSSION The aim of the present work is to develop a facile, general, and efficient approach to obtain advanced intelligent MIPs with both photo- and thermoresponsive template binding properties in aqueous media, because they are highly promising in such applications as smart separation and assays, as well as controlled drug delivery. Recently, we described the synthesis of pure water-compatible MIP microspheres by the first preparation of “living” MIP microspheres with surface-immobilized dithioester groups via reversible addition−fragmentation chain transfer (RAFT) precipitation polymerization (RAFTPP) and their subsequent surface-grafting of hydrophilic polymer layers via surface-initiated RAFT polymerization.36,37 The introduction of hydrophilic polymer layers onto the MIP microspheres proved to significantly improve their surface hydrophilicity, thus leading to their pure water-compatible binding properties. In addition, the controlled surface-grafting of thermoresponsive PNIPAAm brushes onto the MIP microspheres following a similar method also resulted in pure water-compatible MIP microspheres with thermoresponsive binding properties.37 Very recently, we also reported the successful preparation of photoresponsive azo-containing MIP microspheres via traditional precipitation polymerization by using an acetonitrilesoluble azo functional monomer with a pyridine group (i.e., 4((4-methacryloyloxy)phenylazo)pyridine (MAzoPy), Scheme S1). 21 Furthermore, atom transfer radical precipitation polymerization (ATRPP) has proven highly versatile in preparing “living” MIP microspheres in a one-pot approach,31 which allows the direct surface-grafting of functional polymer brushes via surface-initiated ATRP. Inspired by these encouraging results, we attempted to explore the possibility of preparing water-compatible azo-containing MIP microspheres with both photo- and thermoresponsive template binding properties by the first synthesis of “living” azocontaining MIP microspheres via the ATRPP of MAzoPy and a cross-linker in the presence of a template and their subsequent surface grafting of PNIPAAm brushes via surfaceinitiated ATRP. Synthesis and Characterization of “Living” AzoContaining MIP/CP Microspheres with Surface-Immobilized ATRP Initiating Groups. To fulfill our purpose, the “living” azo-containing MIP/CP microspheres with surfaceimmobilized ATRP initiating groups (i.e., alkyl halide groups) were first synthesized via ATRPP. A model noncovalent molecular imprinting system was chosen here to demonstrate the general principle, which utilized 2,4-D, MAzoPy, EGDMA, and acetonitrile as the template, functional monomer, crosslinker, and porogenic solvent, respectively (2,4-D could form hydrogen bonding with the pyridine group of MAzoPy in the reaction system). ATRPP was carried out by using ethyl 2chloropropionate as the initiator and CuCl/Me6TREN as the catalyst with a reactant combination 2,4-D/MAzoPy/EGDMA/ ethyl 2-chloropropionate/CuCl/Me6TREN of 1/1/3/0.035/ 0.035/0.105 (molar ratio) and the volume of the utilized acetonitrile was 99% relative to that of the whole reaction system. The use of Me 6 TREN as the ligand in the polymerization system can be ascribed to its stronger interaction with copper catalyst, which should result in better

Figure 1. SEM images of the azo-containing ungrafted MIP (a)/CP (b) and grafted MIP (c)/CP (d) microspheres. The scale bar is 5 μm in the above images.

showed that spherical azo-containing MIP and CP particles were successfully prepared via ATRPP. The number-average diameters (Dn) of the MIP and CP beads were 2.31 and 2.42 μm, respectively, and their size distribution indices (U) were 1.29 and 1.18, respectively. It is worth mentioning here that the azo-containing MIP/CP spherical particles obtained via ATRPP had much larger diameters than those prepared via traditional radical precipitation polymerization under the similar reactant composition (with their Dn values being 1.33 and 1.28 μm, respectively21), although the monomer loading in the ATRPP system was only half of that in the traditional precipitation polymerization system (it has been demonstrated that the sizes of the polymer microspheres should increase with increasing monomer loadings in precipitation polymerization systems39), which might be due to their different particle formation mechanism, just as observed previously.31 Figure 2 presents the FT-IR spectra of the obtained azocontaining MIP and CP microspheres, from which it can be clearly seen that the MIP and CP microspheres have rather similar chemical structures (Figure 2a and b). The presence of three significant peaks around 1732 (CO stretching), 1252, and 1142 cm−1 (C−O−C stretching) supported the existence of poly(EGDMA) in the obtained MIP and CP microspheres. The characteristic peaks corresponding to the CN stretching (1587 cm−1) and CC stretching (1462 cm−1) from the pyridine rings could also be observed, revealing that poly(MAzoPy) was also present in the MIP and CP microspheres. 9770

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FT-IR was first employed to characterize the grafted azocontaining MIP and CP particles (Figure 2c and d). It can clearly be seen that, in addition to the peaks corresponding to the ungrafted MIP/CP particles, some characteristic peaks of PNIPAAm such as the amide I band (1679 cm−1, CO stretching) and amide II band (1530 cm−1, NH stretching) were also observed in the FT-IR spectra of the azo-containing MIP/CP particles obtained via the surface-initiated ATRP of NIPAAm, further verifying the successful grafting of PNIPAAm brushes. Moreover, the results also demonstrated the presence of rather similar grafting levels for the grafted MIP and CP particles, because the ratios of the peak height for the amide I band (1679 cm−1) from PNIPAAm to that for the CN stretching band from the bonded MAzoPy (1587 cm−1) were quite similar in their FT-IR spectra. The surface hydrophilicity of the ungrafted and grafted azocontaining MIP/CP particles was then accurately evaluated by performing static water contact angle experiments. Figure 3a

Figure 2. FT-IR spectra of the azo-containing ungrafted MIP (a)/CP (b) and grafted MIP (c)/CP (d) microspheres.

Moreover, the presence of small peaks around 1639 cm−1 (corresponding to the CC stretching mode of the methacrylate vinyl groups) suggested that less than 100% of the bonded EGDMA molecules were cross-linked in the MIP and CP microspheres. Synthesis and Characterization of Azo-Containing MIP/CP Microspheres with Surface-Grafted PNIPAAm Brushes. With the above-obtained “living” azo-containing MIP/CP microspheres in hand, we started to prepare azocontaining MIP/CP microspheres with surface-grafted PNIPAAm brushes. It is well-known that surface-initiated ATRP is highly robust in the controlled functionalization of various substrates by the facile surface-grafting of functional polymer brushes,38 which can be performed efficiently at room temperature in polar solvents.39,40 In this work, surface-initiated ATRP of NIPAAm was carried out in isopropanol at room temperature to prepare the azo-containing MIP/CP microspheres with grafted PNIPAAm brushes by using the aboveobtained “living” azo-containing MIP/CP (or unmodified or ungrafted MIP/CP) microspheres as the immobilized initiator and CuCl/CuCl2/Me6TREN as the catalyst. Ten mole percent of CuCl2 relative to CuCl was added into the polymerization solution to improve the controllability of the polymerization system.41,42 In addition, a certain amount of ethyl 2chloropropionate was also added into the reaction system as the sacrificial initiator in order to help evaluate the molecular weights and polydispersities of the grafted polymer brushes.43 The polymerization was performed at 25 °C with stirring for 24 h, and the resulting MIP/CP particles were thoroughly washed with methanol to remove the physically adsorbed PNIPAAm. Weight increases of 7% and 6% were observed for the MIP and CP microspheres after their modification, respectively, revealing the successful grafting of PNIPAAm brushes onto the azocontaining MIP/CP particles. It is important to stress here that the increased weights of the modified MIP/CP particles should mainly stem from the surface-grafted polymer brushes, because the use of isopropanol (which is a poor solvent for the “living” azo-containing MIP/CP particles) in the polymer brushesgrafting processes and the rather high cross-linking densities (about 75%) of the MIP/CP microspheres would prevent them from swelling in the reaction media and only allow the occurrence of surface polymerization, just as reported by Tirelli and co-workers44 and our group.39

Figure 3. Profiles of a water drop on the films of polymer microspheres (a) and their dispersion photographs in pure water (1 mg/mL) at 25 °C after the ultrasonically dispersed solutions settled down for 4 h (b). The samples located from left to right in the above figures are the azo-containing ungrafted MIP (1)/CP (2) and grafted MIP (3)/CP (4) microspheres, respectively.

shows the profiles of a water drop on the ungrafted and grafted azo-containing MIP/CP films (as prepared in the experimental part), from which the static water contact angles were determined to be 129°, 127°, 73°, and 71° for the ungrafted MIP, ungrafted CP, grafted MIP, and grafted CP films, respectively. The results clearly showed that the grafted MIP/ CP films exhibited significantly higher hydrophilicity than the ungrafted ones, which again confirmed the presence of PNIPAAm brushes on the surfaces of the modified azocontaining MIP/CP particles. Surface-grafting of hydrophilic polymer brushes has proven to be highly efficient for improving the dispersion stability of the materials in water.36,37,39 Therefore, it is expected that the azo-containing MIP/CP particles grafted with PNIPAAm brushes should show enhanced dispersion stability in water at ambient temperature. The results shown in Figure 3b (SI Figure S1) indeed support this hypothesis. There was much faster sedimentation for the ungrafted MIP/CP microspheres in water in comparison with the grafted ones, further demonstrat9771

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ing that PNIPAAm brushes have been successfully grafted onto the azo-containing MIP/CP microspheres. On the basis of the above results, it can be concluded that the azo-containing MIP/CP microspheres grafted with PNIPAAm brushes have been readily prepared by using the facile surfaceinitiated ATRP approach. Further studies were then carried out in order to get some quantitative information on the grafted polymer brushes including their molecular weights and molecular weight distributions, as well as the thickness of the polymer brush layers. It has been demonstrated that the molecular weights and polydispersities of the free polymers generated in the surfaceinitiated ATRP systems on the spherical particles due to the addition of sacrificial initiator can be utilized to represent those of the grafted polymer brushes,43,45 while different molecular weights and polydispersities were observed for the free polymers and grafted polymer brushes when the surfaceinitiated ATRP took place from the concave and flat surfaces.38,45,46 Therefore, the free polymers obtained in our study were characterized with GPC, from which the numberaverage molecular weights (Mn,GPC) of the grafted PNIPAAm brushes on the MIP and CP particles were evaluated to be 22 200 and 21 800, respectively, and their PDIs were 1.23. The rather similar molecular weights and relatively low polydispersities of the polymer brushes suggested that the surface-initiated ATRP took place in a well-controlled way. The morphologies, particle sizes, and size distributions of the grafted MIP and CP particles were characterized with SEM, and the results clearly showed that both of them were still separate microspheres (Figure 1c and d). The number-average diameters Dn of the grafted MIP and CP microspheres were determined to be 2.33 and 2.44 μm, respectively, and their size distribution indices U were 1.26 and 1.16, respectively. In comparison with the ungrafted MIP and CP microspheres (whose Dn values were 2.31 and 2.42 μm, respectively), an increase of 20 nm in Dn values was obtained for both the grafted MIP and CP microspheres, from which a layer thickness of 10 nm (i.e., ΔDn/2) could be derived for the grafted PNIPAAm brushes (in the dry state). Equilibrium Binding Properties of the Ungrafted and Grafted Azo-Containing MIP/CP Microspheres both in Acetonitrile and in Pure Water. The equilibrium template binding properties of the ungrafted and grafted azo-containing MIP/CP microspheres in acetonitrile were first studied. As shown in Figure 4a, both the ungrafted and grafted MIPs proved to bind more template than their corresponding CPs, suggesting the presence of selective binding sites in the obtained MIPs. It is noteworthy that both the grafted MIP and CP bound less template than their corresponding ungrafted ones in a range of polymer concentrations, which was verified again the occurrence of obvious surface modification for the grafted MIP/CP microspheres. We then switched the equilibrium binding experiments to the pure aqueous solution system. It has been established that the incompatibility of MIPs in pure aqueous media is mainly due to their hydrophobically driven nonspecific bindings, which depend on the hydrophobicity of the template and the exposed MIP surfaces.7,8 As expected, the specific template bindings of the ungrafted MIP almost completely disappeared in the pure aqueous solution and both the ungrafted MIP and CP exhibited rather high binding capacities (Figure 4b), mainly due to their high surface hydrophobicity. Since grafting PNIPAAm brushes onto the azo-containing MIP microspheres has proven to

Figure 4. (a) Equilibrium bindings of 2,4-D (C = 0.05 mM) on different amounts of the ungrafted (square) and grafted (triangle) azocontaining MIP (filled symbols)/CP (open symbols) microspheres in the dark at 25 °C in acetonitrile (a) and in pure water (b), respectively.

significantly improve their surface hydrophilicity at ambient temperature (as revealed by the contact angle and dispersion stability experiments), it can be envisioned that the MIP microspheres grafted with PNIPAAm brushes should show reduced nonspecific bindings in the pure aqueous solution at ambient temperature, thereby enhancing their water-compatibility. This hypothesis is indeed supported by the experimental results (Figure 4b). In sharp contrast to the ungrafted MIP microspheres, the grafted ones showed obvious specific template bindings in the pure aqueous solution at ambient temperature, thus demonstrating the high efficiency of this controlled hydrophilic polymer brush grafting approach for the preparation of water-compatible MIPs. The binding selectivity of the grafted azo-containing MIP/ CP microspheres was first studied by measuring their competitive binding capacities toward 2,4-D and two structurally related compounds DPAc and POAc in their mixed aqueous solution. It can be seen clearly from Figure 5a that, although the grafted MIP microspheres showed much higher binding capacities toward 2,4-D than toward DPAc and POAc, the grafted CP microspheres also exhibited highest affinity to 2,4-D among the tested analytes (i.e., the nonspecific bindings of the grafted MIP microspheres were much higher toward 2,4-D than toward DPAc and POAc), which makes it inappropriate to evaluate the selectivity (or specificity) of the studied MIP microspheres by directly comparing their binding capacities toward 2,4-D, DPAc, and POAc. In this case, the “imprinting-induced promotion of binding” (IPB) has proven to be a useful parameter for evaluating the MIPs’ selectivity, because the difference in the intrinsic nonspecific bindings of the MIPs toward different analytes is normalized.8,35 The IPB values of the grafted MIP microspheres were determined to be 41.4%, 34.0%, and 34.8% toward 2,4-D, DPAc, and POAc, respectively (SI Table S1), which suggested that the grafted MIP microspheres did not show significant selectivity toward 2,4-D in comparison with DPAc and POAc in the aqueous solution, probably due to the rather similar structures between 2,4-D and DPAc and the smaller size of POAc in comparison 9772

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template binding properties. As can be seen clearly from Figure 6a and b, both the equilibrium binding capacities of the

Figure 5. (a) Selective bindings of the grafted azo-containing MIP (filled columns)/CP (empty columns) microspheres toward 2,4-D, DPAc, and POAc in their mixed solution in pure water (C2,4‑D, DPAc or POAc = 0.05 mM), respectively (polymer concentration: 4 mg/mL). (b) Selective bindings of the grafted azo-containing MIP (filled columns)/CP (empty columns) microspheres toward 2,4-D, 2,4-DCP, and 2,4-DCAD in their mixed solution in pure water (C2,4‑D, 2,4‑DCP or 2,4‑DCAD = 0.05 mM), respectively (polymer concentration: 4 mg/mL).

Figure 6. Equilibrium bindings of 2,4-D (0.05 mM) on different amounts of the ungrafted azo-containing MIP (filled symbols)/CP (open symbols) microspheres in the dark (square) and under UV light irradiation (diamond) in acetonitrile, respectively (a), and those of 2,4D (0.05 mM) on different amounts of the grafted azo-containing MIP (filled symbols)/CP (open symbols) microspheres in the dark (downpointing triangle) and under UV light irradiation (circle) in pure water (at 25 °C), respectively (b).

with 2,4-D (which allows them to enter the 2,4-D imprinted binding sites easily). While the presence of certain crossbinding reactivity in the MIPs might be undesirable for such applications as sensors, this could actually be an advantage in some applications like drug delivery, because a series of analogous compounds could be loaded into the binding sites efficiently. Moreover, the high nonspecific template binding on the studied MIP microspheres is also helpful for their potential application in drug delivery because a higher template loading can be realized. To clearly demonstrate the specificity of the grafted MIP microspheres toward the template and the successful molecular imprinting process, we further studied the binding selectivity of the grafted MIP/CP microspheres by measuring their competitive binding capacities toward 2,4-D and another two structurally related compounds 2,4-DCP and 2,4-DCAD in their mixed aqueous solution. As can be seen from SI Scheme S2, the chemical structures of 2,4-DCP and 2,4-DCAD are rather different from that of 2,4-D in the sense that they do not have a carboxyl group. Therefore, it is expected that the grafted MIP microspheres should show much higher IPB values toward 2,4-D than toward 2,4-DCP and 2,4-DCAD. The experimental results indeed support this hypothesis, and the IPB values of the grafted MIP microspheres proved to be 35.1%, 17.0%, and 13.1% toward 2,4-D, 2,4-DCP, and 2,4-DCAD, respectively (Figure 5b and SI Table S1). Photoresponsive Binding Properties of the AzoContaining MIP/CP Microspheres. The equilibrium binding experiments were then performed by incubating a 2,4-D solution with different amounts of ungrafted or grafted MIP/ CP microspheres at ambient temperature for 6 h under the irradiation of UV light to check whether the obtained azocontaining MIP microspheres could show photoresponsive

ungrafted azo-containing MIP microspheres in acetonitrile and those of the grafted azo-containing MIP microspheres in pure water decreased upon exposure to 365 nm UV light in the range of polymer concentrations, suggesting that the template binding properties of the azo-containing MIP microspheres were indeed photoresponsive (note that the reversible photochemical isomerization behaviors of the grafted azocontaining MIP/CP microspheres in aqueous solution were confirmed prior to their photoresponsive template binding studies (SI Figures S2 and S3)). It is worth noting here that, in contrast with the ungrafted and grafted azo-containing MIP microspheres, a certain increase in the equilibrium binding capacities was observed for their corresponding azo-containing CP microspheres under the irradiation of UV light, just as observed in our previous report.21 The real cause of this phenomenon is not yet fully understood, but it might be ascribed to the increase in the interactions between the CP particles and template molecules because of the polarity change of the azo groups under the UV light irradiation,47 thus leading to their increased nonspecific template binding capacities. To exclude these photoinduced nonspecific template binding changes, the specific template bindings (normally defined as the binding difference between the MIP and its corresponding CP) of the ungrafted and grafted azo-containing MIP microspheres were presented (Figure 7), which demonstrated the photoresponsive template binding properties of the ungrafted and grafted MIP microspheres more clearly. On the basis of the above results, it can be concluded that the trans to cis photoisomerization of the azo chromophores induced by the UV light irradiation can indeed lead to the alteration of the spatial arrangement of the binding functionalities within the 9773

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and its analogues by the grafted MIP/CP in the aqueous solution was also carried out to obtain the photoswitching conditions (SI Figure S4)). The experiments were performed by first incubating a series of samples (which were composed of 2 mg of the grafted azo-containing MIP/CP microspheres and 0.5 mL of a solution containing 2,4-D, DPAc, and POAc in pure water (C2,4‑D, DPAc or POAc = 0.05 mM)) at 25 °C in the dark for 6 h until the equilibrium analyte bindings were reached. As expected, the grafted azo-containing MIP microspheres showed significantly higher binding capacities toward 2,4-D than toward DPAc and POAc. The UV light irradiation on the above mixed solutions with equilibrium analyte bindings led to the obvious release of the template from the grafted azo-containing MIP microspheres into the solutions. The subsequent thermal backisomerization of the above system in the dark caused an increase in the template binding capacities (note that the visible light-induced back-isomerization of the azo-containing MIP microspheres could also be used to cause the template uptake from the solutions). Repeating the photoswitching cycles resulted in the release and uptake of 2,4-D in quantities very similar to those of the previous cycles, which clearly demonstrated the reversibility of the binding site configuration and substrate affinity in the course of photoswitching of azo chromophores. Note that the grafted azo-containing MIP microspheres were also able to bring about some degree of photoregulated release and uptake for the structural analogues of the template (i.e., DPAc and POAc), again suggesting the presence of certain cross-binding reactivity in the grafted azocontaining MIP microspheres. The dependence of the specific analyte bindings of the grafted azo-containing MIP microspheres on photoswitching conditions were also presented (Figure 8b), which demonstrated their photoregulated release and uptake of the template and its analogues more clearly. On the basis of the above results, it can be concluded that the grafted azo-containing MIP microspheres indeed show obvious photoresponsive template binding properties in the pure aqueous solution, and the affinity of the binding sites in the MIP microspheres toward the template can be easily tuned by simple photoswitching. In comparison with the previous report by Lam and co-workers for the preparation of water-compatible photoresponsive MIP hydrogels (where water-soluble azo functional monomers had to be used),13 our strategy allows more efficient synthesis of water-compatible and photoresponsive MIP microspheres with good mechanical properties by directly using widely available water-insoluble azo functional monomers, normal molecular imprinting recipes, and the facile surface-grafting of hydrophilic polymer brushes, which opens up a new and versatile avenue for obtaining such advanced intelligent MIPs. To our knowledge, this is the first demonstration of the synthesis of micrometer-sized spherical azo-containing MIP beads with photoregulated release and uptake capabilities toward the template molecule in pure aqueous media. Thermoresponsive Binding Properties of the Grafted Azo-Containing MIP/CP Microspheres in Aqueous Media. PNIPAAm has been well-known as a thermoresponsive polymer, which can undergo a conformation change between a hydrated (coiled and soluble) and a dehydrated (collapsed and insoluble) state in water around its lower critical solution temperature (LCST ≈ 32 °C).48 Therefore, the azo-containing MIP/CP microspheres grafted with PNIPAAm brushes are expected to be thermoresponsive in the aqueous solution and might show thermoresponsive binding properties. The results

Figure 7. Specific template bindings of different amounts of the ungrafted and grafted azo-containing MIP microspheres under different conditions: the ungrafted azo-containing MIP microspheres in the dark (filled square) and under UV light irradiation (open square) in acetonitrile, as well as the grafted MIP microspheres in the dark (filled triangle) and under UV light irradiation (open triangle) in pure water (at 25 °C), respectively.

binding sites of the MIP microspheres, thus resulting in their affinity changes, as schematically illustrated in Scheme 1. The photoresponsive binding properties of the grafted azocontaining MIP microspheres were further confirmed by their photoregulated release and uptake of the template molecule in the pure aqueous solution. Figure 8a shows the change of the binding capacities of the grafted azo-containing MIP/CP microspheres toward 2,4-D and its structurally related analogues (DPAc and POAc) in pure water under repetitive photoswitching conditions (a detailed study on the timedependent photoregulated release and uptake of the template

Figure 8. Photoregulated release and uptake of 2,4-D (square) and its analogues (POAc (circle), DPAc (triangle)) by the grafted azocontaining polymer microspheres under photoswitching conditions in aqueous media: (a) Change of the bound analyte amounts (percentage values) for both the grafted MIP (filled symbols) and CP (open symbols) microspheres; (b) Change of the specific analyte bindings for the grafted MIP microspheres (polymer concentrations were 4.0 mg/ mL, the initial concentrations of all the analytes were 0.05 mM, and the duration times for the UV-on and UV-off switching period were 3 and 18 h, respectively). 9774

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microspheres with both photo- and thermoresponsive template binding properties in pure aqueous media by the successive application of ATRPP and surface-initiated ATRP. The resulting PNIPAAm brush-grafted azo-containing MIP microspheres showed significantly enhanced surface hydrophilicity at ambient temperature and excellent photo- and thermoresponsive template binding properties in pure aqueous solutions. In view of the versatility of ATRPP in the synthesis of “living” MIP microspheres with different diameters, the controlled nature of surface-initiated ATRP for the preparation of welldefined polymer brushes with adjustable molecular weights, and the easy availability of various hydrophilic and stimuliresponsive functional monomers, we believe the present methodology to represent a promising way to develop advanced intelligent MIP microspheres with water-compatible stimuli-responsive binding properties for a wide range of templates. Furthermore, we also foresee that such spherical azocontaining MIP particles with surface-grafted PNIPAAm brushes should be of tremendous potential in such applications as smart separation, extraction, and assays, as well as intelligent drug delivery and bioanalytical analysis.

presented in Figure 9 (SI Figure S5) indeed show significant influence of the temperature on the binding properties of the

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ASSOCIATED CONTENT

S Supporting Information *

Schemes S1 and S2, Tables S1 and S2, and Figures S1−S5. This material is available free of charge via the Internet at http:// pubs.acs.org/ .

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Figure 9. (a) Equilibrium bindings of 2,4-D on different amounts of the grafted azo-containing MIP (filled symbols)/CP (open symbols) microspheres in its pure aqueous solution (C = 0.05 mM) at 25 (down-pointing triangle) and 45 °C (up-pointing triangle), respectively. (b) Temperature dependence of the specific template bindings of the grafted azo-containing MIP microspheres (polymer concentration: 4.0 mg/mL; C2,4‑D = 0.05 mM).

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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grafted azo-containing MIP microspheres. The specific template bindings of the grafted MIP dramatically decreased at an elevated temperature (45 °C) in the pure aqueous solution in comparison with those observed at ambient temperature (Figure 9a), which is likely to be due to the collapsing of the polymer brushes at the higher temperature, thus resulting in the blocking of the binding sites (Scheme 1), just as reported by the group of Hoffman in a protein system.49 A detailed investigation was further carried out to get more insight into the dependence of the template binding properties on the environmental temperatures (SI Figure S5). It can be seen clearly from Figure 9b that the specific template bindings of the grafted azo-containing MIP decreased dramatically with increasing the temperatures of the aqueous solutions from 25 to 30 °C, suggesting the presence of a LCST there for the grafted PNIPAAm brushes, which agreed well with the previous report.37,50 The above results, together with the experimental findings that the specific template bindings of the ungrafted azo-containing MIP in the aqueous solution and those of the grafted azo-containing MIP in acetonitrile were kept almost unchanged with the change of the temperature (SI Table S2), strongly verified that it is indeed the thermoresponsive conformation change of the PNIPAAm brushes that imparts the grafted azo-containing MIP with thermoresponsive template binding properties.

ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support from National Natural Science Foundation of China (20744003, 20774044, 20974048, 21174067), Natural Science Foundation of Tianjin (11JCYBJC01500), the supporting program for New Century Excellent Talents (Ministry of Education) (NCET-070462), and the project sponsored by SRF for ROCS, SEM ([2008]890).

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REFERENCES

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