Well-Defined Hydrophilic Molecularly Imprinted Polymer Microspheres

Article pubs.acs.org/Biomac

Well-Defined Hydrophilic Molecularly Imprinted Polymer Microspheres for Efficient Molecular Recognition in Real Biological Samples by Facile RAFT Coupling Chemistry Man Zhao, Xiaojing Chen, Hongtao Zhang, Husheng Yan, and Huiqi Zhang* Key Laboratory of Functional Polymer Materials, Ministry of Education, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), and College of Chemistry, Nankai University, Tianjin 300071, P. R. China S Supporting Information *

ABSTRACT: A facile and highly efficient new approach (namely RAFT coupling chemistry) to obtain well-defined hydrophilic molecularly imprinted polymer (MIP) microspheres with excellent specific recognition ability toward small organic analytes in the real, undiluted biological samples is described. It involves the first synthesis of “living” MIP microspheres with surface-bound vinyl and dithioester groups via RAFT precipitation polymerization (RAFTPP) and their subsequent grafting of hydrophilic polymer brushes by the simple coupling reaction of hydrophilic macro-RAFT agents (i.e., hydrophilic polymers with a dithioester end group) with vinyl groups on the “living” MIP particles in the presence of a free radical initiator. The successful grafting of hydrophilic polymer brushes onto the obtained MIP particles was confirmed by SEM, FT-IR, static contact angle and water dispersion studies, elemental analyses, and template binding experiments. Welldefined MIP particles with densely grafted hydrophilic polymer brushes (∼1.8 chains/nm2) of desired chemical structures and molecular weights were readily obtained, which showed significantly improved surface hydrophilicity and could thus function properly in real biological media. The origin of the high grafting densities of the polymer brushes was clarified and the general applicability of the strategy was demonstrated. In particular, the well-defined characteristics of the resulting hydrophilic MIP particles allowed the first systematic study on the effects of various structural parameters of the grafted hydrophilic polymer brushes on their water-compatibility, which is of great importance for rationally designing more advanced real biological samplecompatible MIPs.

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mimicking alcoholic beverages,31,32 and pure water33−41) and in rather dilute biological samples (containing 40 vol% of a mixture of ethanol/water (1:1 v/v) and a phosphate buffer)42 have been developed. However, the design of MIPs that are directly applicable in the real, undiluted biological samples for specifically recognizing the targeted small organic analytes has been a formidable challenge because of the complex nature of the sample matrices.15,16,19 Recently, our group reported the successful synthesis of the first series of MIPs with efficient specific recognition ability toward small organic analytes in the real, undiluted biological samples (including pure milk and bovine serum) by using the one-pot hydrophilic macromolecular chain-transfer agent (macro-CTA)-mediated reversible addition−fragmentation chain transfer (RAFT) precipitation polymerization (RAFTPP),43 which proved to be very attractive synthetic substitutes for biological receptors in bioanalytical applications. However, the addition of hydrophilic macro-CTAs (or macroRAFT agents) into the molecular imprinting polymerization systems was required during the synthesis of such MIPs, which

INTRODUCTION Molecularly imprinted polymers (MIPs) are tailormade synthetic receptors with high affinity and specificity toward the targeted analytes.1−12 They are typically prepared by the first copolymerization of a functional monomer and a crosslinking monomer in the presence of a template molecule and a suitable porogenic solvent and the subsequent removal of the template from the resulting cross-linked polymers. The obtained MIPs have the imprinted binding sites (cavities) complementary to the shape, size, and functionality of the template and can thus specifically recognize the template molecule. This, together with their high stability, ease of preparation, and low cost makes them very promising synthetic substitutes for biological receptors. However, the presently developed MIPs targeting small organic molecules are normally only organic solvent-compatible, and they mostly fail to show specific template bindings in the aqueous solutions, which significantly limits their practical applications in such fields as immunoassay and biomimetic sensors.13−21 Many efforts have been devoted to addressing this issue in the past two decades and some MIPs applicable for the direct detection of small organic analytes in relatively simple aqueous solutions (such as surfactant-containing water,22 aqueous buffer solutions (mostly containing an organic solvent),14,23−30 beer or solutions © 2014 American Chemical Society

Received: January 19, 2014 Revised: March 6, 2014 Published: March 25, 2014 1663

dx.doi.org/10.1021/bm500086e | Biomacromolecules 2014, 15, 1663−1675

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Scheme 1. Chemical Structures of the Functional Monomers (4-VP and MAA), Cross-Linker (EGDMA), Templates (2,4-D and Propranolol), Hydrophilic Macro-CTAs, and RAFT Agent (CDB), as Well as Schematic Protocol for the Synthesis of WellDefined MIP Particles with Densely Grafted Hydrophilic Polymer Brushes by the Facile RAFT Coupling Chemistry and Their Specific Recognition of Small Organic Analytes in the Real, Undiluted Biological Samples

Scheme 1. Well-defined MIP particles with densely grafted hydrophilic polymer brushes (∼1.8 chains/nm2) of desired chemical structures and molecular weights were readily obtained, which could work properly in the real, undiluted biological media due to the presence of the effective hydrophilic protection layers.43,45−47 In comparison to our previously reported one-pot method,43 our new two-step approach has some distinct advantages such as its synthetic flexibility, because RAFTPP allows the facile and controlled one-pot synthesis of “living” MIP particles with high densities of surface-bound vinyl groups and adjustable sizes for a wide range of templates under the normal imprinting conditions and the subsequent simple macro-CTA-mediated coupling reaction can in principle make all of them compatible with real biological samples easily. In addition, the well-defined characteristics of the resulting hydrophilic MIP microspheres make it possible, for the first time, to systematically study the effects of various structural parameters (i.e., chemical structures, molecular weights, and grafting densities) of the grafted hydrophilic polymer brushes on their water compatibility, which provided important information for rationally designing more advanced real biological sample-compatible MIPs. Note that a two-step approach for preparing pure water-compatible MIP particles by the first synthesis of “living” MIP particles via RAFTPP and their surface-grafting of hydrophilic polymer brushes via

might have some negative influences on the noncovalent interactions between the template and the functional monomer and thus on the binding properties of the resulting MIPs. In addition, some important structural parameters of the resulting hydrophilic MIP particles (e.g., the grafting density of the hydrophilic polymer brushes) cannot be obtained due to the complicated nature of the one-pot polymerization, which makes it difficult to get more insight into the detailed structure− property relationships of these MIPs and thus influences their further developments. Therefore, new and versatile approaches that can overcome the above problems are highly desirable for the synthesis of well-defined hydrophilic MIPs compatible with real biological samples. Herein, we report a facile, general, and highly efficient new approach (namely, RAFT coupling chemistry) to obtain welldefined hydrophilic MIP microspheres that show excellent specific molecular recognition ability toward small organic analytes in the real, undiluted biological samples (including the pure milk and bovine serum). It involves the first synthesis of “living” spherical MIP particles with surface-bound vinyl and dithioester groups via RAFTPP12,44 and their subsequent grafting of hydrophilic polymer brushes by the simple coupling reaction of the hydrophilic macro-CTAs with vinyl groups on the “living” MIP particles in the presence of a free radical initiator (e.g., azobisisobutyronitrile, AIBN), as illustrated in 1664


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Table 1. Synthetic Conditions for the Well-Defined MIP/CP Microspheres with Surface-Grafted Hydrophilic Polymer Brushes and Their Characterization Data MIP/CP used for coupling

Macro-CTA used

Mn, Mn,NMRor Mn,GPCb

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

2,4-D-MIP 2,4-D-CP 2,4-D-MIP 2,4-D-CP 2,4-D-MIP 2,4-D-CP 2,4-D-MIP 2,4-D-CP 2,4-D-MIP 2,4-D-CP 2,4-D-MIP 2,4-D-CP 2,4-D-MIP 2,4-D-CP 2,4-D-MIP 2,4-D-CP 2,4-D-MIP 2,4-D-CP 2,4-D-MIP 2,4-D-CP 2,4-D-MIP 2,4-D-CP 2,4-D-MIP 2,4-D-CP 2,4-D-MIP

PEG PEG PEG PEG PEG PEG PEG PEG PNIPAAm PNIPAAm PNIPAAm PNIPAAm PNIPAAm PNIPAAm PNIPAAm PNIPAAm -

1000 1000 2000 2000 5000 5000 10000 10000 2470 2470 6360 6360 10300 10300 14600 14600 27600 29000 35300 37900 42000 43600 -

26 27

2,4-D-CP 2,4-D-CP

PEG

10000

28

2,4-D-CP

PNIPAAm

10300

29 30 31 32 33 34 35 36 37 38

Propranolol-MIP Propranolol-CP Propranolol-MIP Propranolol-CP Propranolol-MIP Propranolol-CP Propranolol-MIP Propranolol-CP Propranolol-MIP Propranolol-CP

PEG PEG PEG PEG PNIPAAm PNIPAAm PNIPAAm PNIPAAm

5000 5000 10000 10000 10300 10300 14600 14600

entrya

(1.25) (1.24) (1.13) (1.13) (1.21) (1.18)

grafting methodc

ΔW (%)d

Dn (μm)e

RAFT coupling RAFT coupling RAFT coupling RAFT coupling RAFT coupling RAFT coupling RAFT coupling RAFT coupling RAFT coupling RAFT coupling RAFT coupling RAFT coupling RAFT coupling RAFT coupling RAFT coupling RAFT coupling SI-RAFT (12 h) SI-RAFT (12 h) SI-RAFT (24 h) SI-RAFT (24 h) SI-RAFT (36 h) SI-RAFT (36 h) -

0.65 0.69 1.29 1.36 3.15 3.35 6.23 6.56 1.61 1.69 4.12 4.35 6.53 6.96 8.97 9.35 7.97 8.96 11.44 12.52 13.52 13.91 -

Coupling reaction Coupling reaction RAFT coupling RAFT coupling RAFT coupling RAFT coupling RAFT coupling RAFT coupling RAFT coupling RAFT coupling

8.60

2.809 2.696 2.811 2.698 2.813 2.700 2.818 2.704 2.825 2.710 2.813 2.699 2.819 2.704 2.824 2.709 2.829 2.713 2.825 2.713 2.831 2.718 2.836 2.724 Particle aggregate 1.153 1.163

contact angle (o)h

βf

Ue 1.028 1.021 1.023 1.024 1.027 1.022 1.026 1.023 1.024 1.022 1.024 1.020 1.026 1.023 1.031 1.019 1.024 1.030 1.029 1.023 1.028 1.023 1.020 1.022

1.83 1.87 1.82 1.84 1.78 1.82 1.77 1.78 1.84 1.85 1.83 1.85 1.80 1.84 (1.84) 1.75 1.75 0.82 0.84 0.92 0.90 0.92 0.89 -

1.017 1.018

1.00

9.19

1.164

1.015

1.04 (0.96)

3.04 2.49 5.69 4.88 6.09 5.18 8.49 7.08

3.045 3.577 3.051 3.584 3.057 3.589 3.059 3.589 3.062 3.593

1.021 1.025 1.016 1.022 1.020 1.033 1.019 1.031 1.029 1.034

1.86 1.79 1.75 1.76 1.81 1.81 1.79 1.75

g

121.8 122.6 88.9 91.3 71.6 72.3 65.4 66.0 64.3 65.4 77.1 78.3 72.1 73.2 66.5 67.5 68.2 67.0 79.6 80.5 67.4 70.6 65.0 65.4 124.9

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.8 1.4 1.5 1.0 1.7 1.3 1.5 1.4 1.8 1.0 1.3 1.6 1.2 1.0 1.1 1.0 1.0 1.1 1.4 1.6 1.5 1.8 1.3 1.0 1.9

125.1 ± 1.1 88.8 ± 1.5 g

87.9 ± 1.6 122.6 121.6 70.5 69.8 66.9 65.6 66.7 68.5 70.1 68.8

± ± ± ± ± ± ± ± ± ±

1.0 1.6 1.0 1.4 2.1 1.8 1.5 1.0 1.9 1.4

Entries 1 and 2 are the ungrafted “living” 2,4-D-MIP and 2,4-D-CP with surface dithioester and vinyl groups prepared via RAFTPP, respectively; entries 3−18 the 2,4-D-MIPs/2,4-D-CPs bearing hydrophilic brushes prepared via the coupling reactions between entry 1 or 2 with macro-CTAs; entries 19−24 the 2,4-D-MIPs/2,4-D-CPs bearing PNIPAAm brushes prepared via the surface-initiated RAFT polymerization of NIPAAm with entry 1 or 2 as the immobilized RAFT agent; entries 25 and 26 the ungrafted 2,4-D-MIP and 2,4-D-CP prepared via TRPP under the same condition as RAFTPP but in the absence of CDB, respectively; entries 27 and 28 the 2,4-D-CPs with hydrophilic brushes prepared via the coupling reaction of entry 26 with macro-CTAs; entries 29 and 30 the ungrafted “living” propranolol-MIP and propranolol-CP with surface dithioester and vinyl groups prepared via RAFTPP, respectively; and entries 31−38 the propranolol-MIPs/propranolol-CPs bearing hydrophilic brushes prepared via the coupling reaction of entry 29 or 30 with macro-CTAs. bMn denotes the molecular weight of PEG in PEG macro-CTAs for entries 3−10, 27, and 31− 34; Mn,NMR the number-average molecular weight of PNIPAAm macro-CTA (for entries 11−18, 28, and 35−38) determined by 1H NMR; and Mn,GPC the number-average molecular weight of PNIPAAm brushes for entries 19−24 determined by GPC (the data inside the brackets are the dispersities of the polymer brushes). c“RAFT coupling” refers to the coupling reaction between the macro-CTAs and ungrafted “living” MIP/CP particles and “SI-RAFT” the surface-initiated RAFT polymerization (the data inside the brackets are the grafting times). dWeight increases (ΔW (%)) of the grafted MIPs/CPs are expressed as the percentage values (i.e., the increased weights relative to the original weights). eDn and U refer to the number-average diameter and size distribution index of the MIP/CP microspheres, respectively, as determined by SEM. fβ (chains/nm2) is the grafting density of the polymer brushes determined by the measured weight increase of the grafted polymer microspheres unless otherwise mentioned. gData inside the brackets are the grafting densities of the polymer brushes determined by elemental analyses. hStatic water contact angles of the polymer films. a

particles bearing hydrophilic polymer brushes with much lower grafting densities (∼1 chain/nm2) compared with our new

surface-initiated RAFT polymerization of hydrophilic monomers was also described.33,34 However, it only led to MIP 1665


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vinyl groups were synthesized via RAFTPP according to our previously reported method,33 but with some modification in the reactant composition and experimental procedure as follows: 4-VP (80 μL, 0.75 mmol), 2,4-D (41.56 mg, 0.188 mmol), and a mixture of methanol and water (4:1 v/v, 60 mL) were added into a one-neck round-bottom flask (100 mL) successively. A clear solution was obtained after 30 min of stirring at room temperature, to which EGDMA (0.71 mL, 3.76 mmol), CDB (45.12 mg, 0.166 mmol), and AIBN (13.61 mg, 0.083 mmol) were added. After being purged with argon for 30 min, the reaction mixture was sealed and stirred at 25 °C for another 2 h in order to allow the self-assembly of the functional monomer and template. The reaction flask was then attached to the rotor-arm of a rotary evaporator, immersed into a thermostatted oil bath at 60 °C, and rotated slowly (ca. 20 rpm) for 24 h. The resulting polymer particles in the reaction solutions were collected by centrifugation and then washed thoroughly with methanol/acetic acid (9:1 v/v) and methanol successively until no template could be detected in the washing solution. After being dried at 40 °C under vacuum for 48 h, a light pink MIP was obtained in a yield of 65% (entry 1 in Table 1). The above polymerization was then repeated twice and all the obtained 2,4-D-MIP microspheres were mixed together for further study. The “living” 2,4-D-CP microspheres with surface-bound dithioester and vinyl groups were prepared and purified under the identical conditions except that the template was omitted (yield: 69%) (entry 2 in Table 1). Synthesis of the “Living” Propranolol-Imprinted Polymer (i.e., Propranolol-MIP)/Propranolol-CP Microspheres with Surface-Bound Dithioester and Vinyl Groups via RAFTPP. The “living” propranolol-MIP microspheres with surface-bound dithioester and vinyl groups were synthesized via RAFTPP according to the following procedure: MAA (84.7 μL, 1 mmol), propranolol (86.30 mg, 0.33 mmol), and a mixture of acetonitrile and methanol (9:1 v/v, 60 mL) were added into a one-neck round-bottom flask (100 mL) successively. A clear solution was obtained after 30 min of stirring at room temperature, to which EGDMA (0.94 mL, 5 mmol), CDB (60.01 mg, 0.22 mmol), and AIBN (18.04 mg, 0.11 mmol) were added. After being purged with argon for 30 min, the reaction mixture was sealed and stirred at 25 °C for another 2 h in order to allow the self-assembly of the functional monomer and template. The reaction flask was then attached to the rotor-arm of a rotary evaporator, immersed into a thermostatted oil bath at 60 °C, and rotated slowly (ca. 20 rpm) for 24 h. The resulting polymer particles were collected by centrifugation and then washed thoroughly with methanol/acetic acid (9:1 v/v) and methanol successively until no template could be detected in the washing solution. After being dried at 40 °C under vacuum for 48 h, a light pink MIP was obtained in a yield of 74% (entry 29 in Table 1). The “living” propranolol-CP microspheres with surface-bound dithioester and vinyl groups were prepared and purified under the identical conditions except that the template was omitted (yield: 79%) (entry 30 in Table 1). Synthesis of the 2,4-D-MIP/2,4-D-CP Microspheres with Surface-Grafted PEG or PNIPAAm Brushes via the Coupling Reaction between the Hydrophilic Macro-CTAs and “Living” 2,4-D-MIP/2,4-D-CP Microspheres Prepared via RAFTPP. The 2,4-D-MIP/2,4-D-CP microspheres with surface-grafted hydrophilic polymer brushes were prepared by the coupling reaction between the hydrophilic macro-CTAs and “living” 2,4-D-MIP/2,4-D-CP microspheres with surface-bound dithioester and vinyl groups in the presence of a free radical initiator. A typical procedure for the synthesis of 2,4-D-MIP/2,4-D-CP microspheres with PEG brushes (Mn = 5000) is presented as follows: the “living” 2,4-D-MIP/2,4-D-CP microspheres with surface-bound dithioester and vinyl groups (150 mg), PEG macro-CTA (Mn,PEG = 5000) (0.0736 mmol), AIBN (0.0238 mmol), and DMF (30 mL) were added into a one-neck eggplantshaped flask (50 mL) successively. After being degassed with five freeze−pump−thaw cycles, the reaction mixture was sealed and then immersed into a thermostatted oil bath at 60 °C. The coupling reaction was allowed to take place for 48 h under stirring. The

strategy (as also confirmed in the present study), which largely influences the water-compatibility of the obtained MIPs. In addition, although recent years have witnessed considerable interest in the surface modification of polymer microspheres with polymer brushes, and some efficient coupling approaches have been developed for this purpose, such as copper-catalyzed Huisgen 1,3-dipolar cycloaddition of alkynes and azides48,49 and thiol−ene chemistry,48−50 to our knowledge, the application of this RAFT coupling chemistry in the synthesis of functionalized MIP/polymer particles has never been reported. Furthermore, the rather high grafting densities of polymer brushes achieved for the modified polymer/MIP microspheres by our approach and its direct use of macro-CTAs instead of thiol groupterminated polymers (which are used in the thiol−ene chemistry and are normally obtained by thiol-modification of macro-CTAs prepared via RAFT polymerization48−50) makes it highly applicable.

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EXPERIMENTAL SECTION

Materials. 4-Vinylpyridine (4-VP, Alfa Aesar, 96%), methacrylic acid (MAA, Tianjin Jiangtian Chemicals, 99%), and ethylene glycol dimethacrylate (EGDMA, Alfa Aesar, 98%) were purified by distillation under vacuum. N,N-Dimethylformamide (DMF, Tianjin Jiangtian Chemicals, 99.5%) was dried with anhydrous magnesium sulfate and then distilled under vacuum. Methanol (MeOH, Tianjin Jiangtian Chemicals, Analytical grade (AR)) was distilled prior to use. Acetonitrile (ACN, Tianjin Kangkede Chemicals, AR), toluene (Tianjin Jiangtian Chemicals, AR), and dichloromethane (Tianjin Jiangtian Chemicals, AR) were refluxed over calcium hydride and then distilled. Dioxane (Tianjin Jiangtian Chemicals, AR) was refluxed with sodium and then distilled before use. Azobisisobutyronitrile (AIBN, Chemical Plant of Nankai University, AR) was recrystallized from ethanol. N-Isopropylacrylamide (NIPAAm, Acros, 99%) was recrystallized from hexane. (±)-Propranolol hydrochloride (Alfa Aesar, 99%) was converted into its free base form (Scheme 1) before use following the previously reported procedure.51 Atenolol (National Institute for the Control of Pharmaceutical and Biological Products, China, Chemical reference substance, Scheme S1) was dried at 105 °C for 3 h before use. Cumyl dithiobenzoate (CDB, Scheme 1) was prepared following a literature procedure.52 2-(Phenylcarbonothioylthio)butanoic acid was synthesized according to the literature method.53 A series of well-defined poly(NIPAAm) (PNIPAAm) with a dithioester end group (i.e., PNIPAAm macro-CTAs, Scheme 1) were synthesized via the RAFT polymerization of NIPAAm following the literature method (Figure S1, Table S1).54 A series of PEG macroCTAs (Scheme 1) were prepared by the reaction of monomethoxycapped PEG (with an average molecular weight Mn = 1000, 2000, 5000, or 10000) with 2-(phenylcarbonothioylthio) butanoic acid following our previous approach.35 The pure milk (Inner Mongolia Yili Industrial Group) was purchased from a local supermarket, which was centrifuged at 10000 rpm for 15 min in order to remove the possibly existed particles inside the milk, and the homogenously suspended milk was used for the binding analyses. The standard fetal bovine serum (Beijing Solarbio Science & Technology Co., Ltd.) was stored at −20 °C prior to use and the thawed bovine serum was directly used in our study. 2,4-Dichlorophenoxyacetic acid (2,4-D, Alfa Aesar, 98%, Scheme 1), phenoxyacetic acid (POAc, Acros, 98+%, Scheme S1), monomethoxy-capped poly(ethylene glycol) (PEG) (abbreviated as PEG) with an average molecular weight (Mn) of 1000, 2000, 5000, and 10000 (Aldrich), N,N′-dicyclohexylcarbodiimide (DCC, Tianjin Jiangtian Chemicals, AR), 4-(dimethylamino)pyridine (DMAP, Merck, AR), and all the other reagents were commercially available and used as received unless otherwise stated. Synthesis of the “Living” 2,4-D-Imprinted Polymer (i.e., 2,4D-MIP)/Nonimprinted Polymer or Control Polymer (2,4-D-CP) Microspheres with Surface-Bound Dithioester and Vinyl Groups via RAFT Precipitation Polymerization (RAFTPP). The “living” 2,4-D-MIP microspheres with surface-bound dithioester and 1666


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resulting polymer particles were collected by centrifugation and thoroughly washed with methanol, and then dried at 40 °C under vacuum to constant weight, leading to 2,4-D-MIP and 2,4-D-CP microspheres bearing PEG brushes (Mn = 5000) with a weight of 154.73 and 155.03 mg, respectively (entries 7 and 8 in Table 1). The 2,4-D-MIP/2,4-D-CP microspheres with surface-grafted PEG brushes of different molecular weights (Mn = 1000, 2000, and 10000) (entries 3−6, 9, and 10 in Table 1) and those with PNIPAAm brushes (Mn,NMR = 2470, 6360, 10300, and 14600) (entries 11−18 in Table 1) were also prepared following the same reactant composition and experimental procedure. Synthesis of Propranolol-MIP/Propranolol-CP Microspheres with Surface-Grafted PEG or PNIPAAm Brushes via the Coupling Reaction between the Hydrophilic Macro-CTAs and “Living” Propranolol-MIP/Propranolol-CP Microspheres Prepared via RAFTPP. The propranolol-MIP/propranolol-CP microspheres with surface-grafted PEG (Mn = 5000 and 10000) or PNIPAAm (Mn,NMR = 10300 and 14600) brushes were prepared following the procedure described in the section Synthesis of the 2,4D-MIP/2,4-D-CP Microspheres with Surface-Grafted PEG or PNIPAAm Brushes via the Coupling Reaction between the Hydrophilic Macro-CTAs and “Living” 2,4-D-MIP/2,4-D-CP Microspheres Prepared via RAFTPP except that the “living” propranolol-MIP/ propranolol-CP microspheres with surface-bound dithioester and vinyl groups were used to replace the “living” 2,4-D-MIP/2,4-D-CP microspheres and the amount of AIBN used was doubled in the coupling reactions (entries 31−38 in Table 1). Synthesis of the 2,4-D-MIP/2,4-D-CP Particles via Traditional Radical Precipitation Polymerization (TRPP). The 2,4-D-MIP/ 2,4-D-CP particles with only surface-bound vinyl groups were prepared by TRPP following the same procedure as the abovedescribed RAFTPP except that CDB was omitted. White MIP and CP were obtained with their yields being 86% and 88%, respectively (entries 25 and 26 in Table 1). Synthesis of the 2,4-D-CP Microspheres with SurfaceGrafted Hydrophilic Polymer Brushes via the Coupling Reaction between the Hydrophilic Macro-CTAs and 2,4-D-CP Microspheres Prepared via TRPP. The 2,4-D-CP microspheres prepared via TRPP were also successfully grafted with PEG (Mn = 10000) or PNIPAAm (Mn,NMR = 10300) brushes by using macroCTA-mediated coupling reaction following the approach described in the section Synthesis of the 2,4-D-MIP/2,4-D-CP Microspheres with Surface-Grafted PEG or PNIPAAm Brushes via the Coupling Reaction between the Hydrophilic Macro-CTAs and “Living” 2,4-D-MIP/2,4D-CP Microspheres Prepared via RAFTPP (entries 27 and 28 in Table 1). Synthesis of the 2,4-D-MIP/2,4-D-CP Microspheres with Surface-Grafted PNIPAAm Brushes via Surface-Initiated RAFT Polymerization (or SI-RAFT). A series of 2,4-D-MIP/2,4-D-CP microspheres with surface-grafted PNIPAAm brushes were also prepared via surface-initiated RAFT polymerization by using the above-obtained “living” 2,4-D-MIP/2,4-D-CP microspheres with surface-bound dithioester and vinyl groups as the immobilized RAFT agent (note that the surface-bound dithioester groups on the “living” 2,4-D-MIP/2,4-D-CP microspheres were used as the grafting points for the surface-initiated RAFT polymerization in this case) in the presence of certain amount of sacrificial CDB following our previously described approach.33 The detailed polymerization procedure is as follows: the “living” 2,4-D-MIP or 2,4-D-CP microspheres with dithioester groups (100 mg), NIPAAm (2.04 g, 18.0 mmol), CDB (4.93 mg, 0.018 mmol), AIBN (1.00 mg, 0.006 mmol), and DMF (5 mL) were added into a one-neck eggplantshaped flask (25 mL) successively. After being degassed with five freeze−pump−thaw cycles, the flask was sealed and immersed into a thermostatted oil bath at 70 °C and stirred for a prescribed time (t = 12, 24, and 36 h). The resulting polymer particles were collected by centrifugation and thoroughly washed with methanol, which were then dried at 40 °C under vacuum to the constant weights, leading to the 2,4-D-MIP microspheres bearing PNIPAAm brushes with a weight of 107.97, 111.44, and 113.52 mg for a polymerization time of 12, 24, and 36 h, respectively (entries 19, 21, and 23 in Table 1) or 2,4-D-CP

microspheres bearing PNIPAAm brushes with a weight of 108.96, 112.52, and 113.91 mg for a polymerization time of 12, 24, and 36 h, respectively (entries 20, 22, and 24 in Table 1). The addition of some sacrificial CDB 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 (after the centrifugation of the reaction mixtures) into ethyl ether, filtered, and then dried at 40 °C under vacuum for 48 h. Characterization. 1H NMR spectra were recorded on a Varian Unity plus-400 spectrometer (400 MHz). A Bio-Rad FTS6000 FT-IR spectrometer was utilized to carry out the FT-IR measurements. The molecular weights and molar-mass dispersities (Đ) of the polymers were determined with a GPC equipped with a Waters 717 autosampler, a Waters 1525 HPLC pump, three Waters UltraStyragel columns (with 5000−600K, 500−30K, and 100−10K molecular ranges) (the temperature of the column oven was 35 °C), and a Waters 2414 refractive index detector. Tetrahydrofuran (THF) (Tianjin Kangkede Chemicals, Chromatographic purity) was used as the eluent at a flow rate of 1.0 mL/min. The calibration curve was obtained by polystyrene (PS) standards. The morphologies, particle sizes, and size distributions of the ungrafted and grafted MIP/CP microspheres were characterized with a scanning electron microscope (SEM, FEI Nova Nano 230). All of the SEM size data reflect the averages of about 200 particles, which are calculated by using the following formulas (Table 1): k

Dn =

k

∑ niDi /∑ ni i=1

i=1

k

Dw =

k

∑ niDi 4 /∑ niDi 3 i=1

U = Dw /Dn

i=1

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 number of the microspheres with a diameter Di. The static water contact angles of the films prepared with MIP/CP microspheres were determined as follows: The 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 and the resulting films were dried at 25 °C under vacuum overnight, a Krüss FM40 Easy Drop contact angle equipment (Germany) was utilized to determine their static water contact angles. Several measurements were taken across each sample, with their average being used for analysis. All the obtained results are listed in Table 1. The dispersion properties of the ungrafted 2,4-D-MIP/2,4-D-CP microspheres and some representative grafted 2,4-D-MIP/2,4-D-CP microspheres in pure water were studied. After their ultrasonic dispersion in pure water (1 mg/mL), the dispersed mixtures were allowed to settle down for different times at 25 °C and their photographs were taken following the time. The elemental analyses of the samples were carried out by using Elementar Vario EL (Germany). The densities of the surface vinyl groups on the “living” 2,4-D-MIP/ 2,4-D-CP microspheres prepared via RAFTPP and that on the 2,4-DCP microspheres prepared via TRPP were obtained by the first saturation of the surface vinyl groups on these polymer microspheres through their addition reactions with bromine following the conditions typical for double bond halogenations55 and the subsequent determination of bromide contents in the resulting brominated polymer particles. A typical procedure is presented as follows: the “living” 2,4-D-MIP microspheres (100 mg) were mixed with 10 mL of chloroform containing 20 mg of bromine and the obtained solution was stirred at ambient temperature for 24 h. After the resulting mixture was washed with an aqueous solution of sodium hydroxide (0.05 M, twice) and water (twice) successively, triethylamine (100 μL) was added into the solution and the mixture was stirred for 12 h at ambient temperature to remove the possible bromide complexed with the pyridine groups on the surfaces of the polymer microspheres. The above mixture was washed with water twice and the solvent 1667


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Figure 1. SEM images of the ungrafted “living” 2,4-D-MIP (a)/2,4-D-CP (d) microspheres prepared via RAFTPP and their grafted ones including the grafted 2,4-D-MIP (b)/2,4-D-CP (e) microspheres with PEG brushes (Mn = 5000) and grafted 2,4-D-MIP (c)/2,4-D-CP (f) microspheres with PNIPAAm brushes (Mn,NMR = 10300) (scale bar is 10 μm). chloroform was then removed under vacuum. The obtained polymer particles were thoroughly washed with DMF and methanol successively, and then dried at 40 °C under vacuum to the constant weights. The “living” 2,4-D-CP microspheres prepared via RAFTPP and 2,4-D-CP microspheres prepared via TRPP were treated similarly. The bromide contents of the resulting brominated MIP/CP microspheres were determined by using the oxygen flask combustion method as shown below:56 the brominated MIP/CP microspheres were first combusted with excessive oxygen in a quartz flask and the resulting gases were absorbed by the deionized water. The bromide ion contents in the solutions were then measured with ion chromatography on an ICS-900 high performance liquid chromatography (Dionex Co., USA) with an electrical conductivity detector (ELCD), from which the bromide contents of the resulting brominated MIP/CP microspheres could be derived. The detailed equilibrium template binding and competitive binding experiments with the ungrafted and grafted MIPs/CPs in different media are described in the Supporting Information.

To evaluate the scope of our strategy and demonstrate its general applicability, a series of well-defined hydrophilic macroCTAs with different chemical structures (i.e., PEG and PNIPAAm macro-CTAs, Scheme 1) and a range of molecular weights (Mn) were first prepared either by the end-group modification of the hydrophilic monomethoxy-capped PEG (M n = 1000, 2000, 5000, and 10000) with 2(phenylcarbonothioylthio)butanoic acid or by the RAFT polymerization of NIPAAm54 (Table S1). They were then allowed to couple with the vinyl groups of the above-obtained “living” 2,4-D-MIP/2,4-D-CP microspheres in the presence of a certain amount of AIBN under the mild reaction conditions (note that it is actually the hydrophilic macroradicals formed by the addition of free radicals (generated from AIBN) to the hydrophilic macro-CTAs that couple with the vinyl groups on the “living” MIP/CP particles), resulting in a series of 2,4-DMIPs/2,4-D-CPs with surface-grafted hydrophilic polymer brushes (entries 3−18 in Table 1). The weights of the MIP and CP microspheres increased after their surface modification, revealing the successful grafting of hydrophilic polymer brushes onto the MIP/CP particles. SEM was first utilized to characterize the above-obtained 2,4D-MIPs/2,4-D-CPs. Both the ungrafted and grafted 2,4-DMIPs/2,4-D-CPs proved to be narrowly dispersed microspheres with their number-average diameters (Dn) being around 2.696−2.829 μm and size distribution indices being around 1.020−1.031 (Figure 1, entries 1−18 in Table 1). In addition, the grafted MIP/CP microspheres showed somewhat larger diameters than their corresponding ungrafted ones (Table 1), suggesting the successful grafting of hydrophilic polymer brushes on the MIP/CP microspheres. The obtained ungrafted and grafted 2,4-D-MIPs/2,4-D-CPs were then characterized with FT-IR (Figure 2a). The presence of three significant peaks around 1730 (CO stretching), 1250, and 1155 cm−1 (C−O−C stretching) supports the existence of poly(EGDMA) in both the ungrafted and grafted MIPs/CPs. The characteristic peaks corresponding to the C N stretching (1595 and 1558 cm−1) and CC stretching (1453 cm−1) in the pyridine rings can also be observed in the spectra of these MIPs/CPs, revealing that poly(4-VP) is also present in these polymers. In addition to the peaks

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RESULTS AND DISCUSSION Synthesis of Well-Defined 2,4-D-MIP/2,4-D-CP Microspheres with Densely Grafted Hydrophilic Polymer Brushes via RAFT Coupling Chemistry and Their Characterization. RAFTPP has proven highly versatile for the facile and controlled one-pot synthesis of “living” MIP microspheres with surface-bound dithioester and vinyl groups, which are very useful precursors for various advanced MIP materials.12 In this work, RAFTPP was first used to prepare “living” 2,4-D-MIP/2,4-D-CP microspheres with surface-bound dithioester and vinyl groups to show proof-of-principle for our strategy. A model noncovalent molecular imprinting system was chosen here, which utilized 2,4-D (a herbcide), 4-VP, EGDMA, and a mixture of methanol and water (4:1 v/v) as the template, functional monomer, cross-linker, and porogenic solvent, respectively. RAFTPP was then performed with AIBN as the initiator and CDB as the RAFT agent in the presence of a large amount of porogenic solvent (about 98% of the total reaction volumes), leading to the light pink “living” 2,4-D-MIP particles with surface-bound dithioester and vinyl groups (entry 1 in Table 1).12,44 The corresponding “living” control polymer (CP) particles were also prepared similarly but in the absence of the template (entry 2 in Table 1). 1668


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polymer brushes should show much reduced static water contact angles and enhanced dispersion stability in water. The experimental results indeed supported this hypothesis (Figure 3, Figure S3, Table 1), which provided strong evidence for the

Figure 3. Profiles of a water drop on the films of the ungrafted and grafted 2,4-D-MIPs/2,4-D-CPs (a) and their dispersion photographs in pure water (1 mg/mL) at 25 °C after settling down for 2 h (b). The samples located from left to right in the above two figures are the ungrafted “living” 2,4-D-MIP (1)/2,4-D-CP (2) prepared via RAFTPP and their grafted ones including the grafted 2,4-D-MIP (3)/2,4-D-CP (4) with PEG brushes (Mn = 5000) and grafted 2,4-D-MIP (5)/2,4-DCP (6) with PNIPAAm brushes (Mn,NMR = 10300).

Figure 2. (a) FT-IR spectra of the ungrafted “living” 2,4-D-MIP (a1)/ 2,4-D-CP (a2) microspheres prepared via RAFTPP and their grafted ones including the grafted 2,4-D-MIP (a3)/2,4-D-CP (a4) microspheres bearing PEG brushes (Mn = 5000) and grafted 2,4-D-MIP (a5)/2,4-D-CP (a6) microspheres bearing PNIPAAm brushes (Mn,NMR = 10300). (b) FT-IR spectra of the ungrafted 2,4-D-CP microspheres prepared via TRPP (b1) and their grafted ones including the grafted 2,4-D-CP microspheres bearing PEG brushes (Mn = 10000) (b2) and grafted 2,4-D-CP microspheres bearing PNIPAAm brushes (Mn,NMR = 10300) (b3) (spectra are positioned in a row on a virtual third axis).

presence of hydrophilic polymer shells on the modified MIP/ CP microspheres. In addition, the static water contact angles of the grafted MIP/CP films were found to essentially decrease with increasing molecular weights of the hydrophilic polymer brushes, which could be ascribed to the better covering of hydrophilic polymer brushes on the MIP/CP particles due to the increase in the chain length of the hydrophilic polymers. Furthermore, the results also clearly showed that the grafted MIPs exhibited rather similar hydrophilicity as their corresponding CP ones, as revealed by their close static water contact angles and dispersion stability in water. By assuming the homogeneous grafting of hydrophilic polymer brushes on the modified 2,4-D-MIP/2,4-D-CP microspheres and an average density ρ = 1 g/cm3 for the ungrafted MIP/CP particle cores, the average surface grafting densities of the hydrophilic polymer brushes (β) can be estimated by using the following equation:33

corresponding to the ungrafted MIP/CP, the characteristic peaks of amide I band (around 1680 cm−1, CO stretching) were also discernible in the FT-IR spectra of the grafted MIPs/ CPs prepared by the coupling reaction between PNIPAAm macro-CTAs and “living” MIP/CP particles (Figure 2a5,a6), verifying the successful grafting of PNIPAAm brushes (note that the characteristic FT-IR absorption peaks of PEG chains were overlapped with those of poly(4-VP-co-EGDMA) cores in the spectra of the grafted MIP/CP microspheres with PEG brushes). Furthermore, there existed a small peak around 1638 cm−1 in the FT-IR spectra of the ungrafted “living” 2,4-D-MIP/ 2,4-D-CP prepared via RAFTPP, which could be ascribed to the CC stretching mode of the residual vinyl groups and demonstrated that less than 100% of the bonded EGDMA molecules were cross-linked in these MIPs/CPs. Note that this peak was still discernible in the grafted MIP/CP microspheres prepared via the coupling reactions, mainly because the amounts of the reacted surface vinyl groups were only a small part of the total amounts of the vinyl groups in the MIP/ CP microspheres (including both the surface vinyl groups and those inside the MIP/CP particles). It has been well demonstrated that surface-grafting of hydrophilic polymer brushes is highly efficient for improving the surface hydrophilicity and water dispersion stability of the polymer particles.33−35 Therefore, it is anticipated that the 2,4D-MIP/2,4-D-CP microspheres grafted with hydrophilic

ΔW (%) = (S × β × M polymer brush)/(NA × V × ρ)

where ΔW refers to the increased weight percentage for the modified MIP/CP due to the surface-grafted polymer brushes (i.e., the increased weight relative to the original weight, Table 1), Mpolymer brush the number-average molecular weight of the grafted PNIPAAm brush determined by 1H NMR (i.e., Mn,NMR, Figure S1, Table 1) or the average molecular weight of PEG brush, NA the Avogadro constant, S the average surface area of the ungrafted MIP/CP particle core [S = 4π(Dn/2)2 is utilized here because the specific surface areas of the ungrafted MIP/ CP microspheres were found to be too small to be accurately determined by nitrogen sorption porosimetry, suggesting their rather smooth surfaces], and V the average volume of the ungrafted MIP/CP particle core [V = (4/3) × π(Dn/2)3]. The average surface grafting densities of the hydrophilic polymer brushes on the above-obtained grafted 2,4-D-MIP/2,4-D-CP particles were evaluated to be about 1.8 chains/nm2 (entries 3− 1669


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particles could thus be derived by using the same equation as shown above (i.e., ΔW(%) = (S × β × Mpolymer brush)/(NA × V × ρ)), which proved to be 1.84 and 0.96 chains/nm2 for the sample entries 16 and 28, respectively (Table 1). These results are in excellent agreement with those derived by using the directly obtained increased weight percentages of the polymer microspheres after their surface modification. In addition, the cross-linking degrees (i.e., bonded EGDMA/(bonded EGDMA + bonded 4-VP), molar ratio) of the ungrafted “living” 2,4-DCP particles prepared via RAFTPP (entry 2 in Table 1) and ungrafted 2,4-D-CP particles prepared via TRPP (entry 26 in Table 1) were also obtained by using both their nitrogen and carbon contents (the carbon contents were 58.68% and 60.31% for the sample entries 2 and 26, respectively, as determined by the elemental analyses) following a literature approach,58 which were 83% and 82%, respectively. To get more insight into the RAFT coupling chemistry and shed light on the origin of the obviously higher grafting densities of hydrophilic polymer brushes on the grafted 2,4-DMIP/2,4-D-CP microspheres prepared via the combined use of RAFTPP and macro-CTA-mediated coupling reaction (i.e., RAFT coupling chemistry) in comparison with those prepared via the combined use of TRPP and macro-CTA-mediated coupling reaction, we tried to measure the densities of surface vinyl groups on the “living” 2,4-D-MIP/2,4-D-CP microspheres prepared via RAFTPP (entries 1 and 2 in Table 1) and that on the 2,4-D-CP microspheres prepared via TRPP (entry 26 in Table 1) by the bromination of the MIP/CP microspheres through the addition reactions between their surface vinyl groups and bromine and the subsequent determination of bromide contents in the resulting brominated MIP/CP microspheres by using the oxygen flask combustion method.56 The bromide contents in the brominated “living” 2,4-D-MIP and 2,4-D-CP microspheres were determined to be 0.082 and 0.095 mmol/g, respectively (note that the high cross-linking degrees of the ungrafted polymer microspheres (82−83% as determined by the elemental analyses) would allow the bromination reactions to take place mainly on their surfaces, just as reported previously for the surface modification of the “living” polymer microspheres with high cross-linking densities59), from which a density of 0.041 and 0.048 mmol surface vinyl groups/g or 11.52 and 12.86 surface vinyl groups/nm2 was derived for the “living” 2,4-D-MIP and 2,4-D-CP microspheres, respectively (by assuming an average density ρ = 1 g/cm3, S = 4π(Dn/2)2, and V = (4/3) × π(Dn/2)3 for the “living” 2,4-D-MIP/2,4-D-CP microspheres). On the other hand, the bromide content in the brominated 2,4-D-CP microspheres (prepared via TRPP) was evaluated to be 0.092 mmol/g, which corresponds to a density of 0.046 mmol vinyl groups/g or 5.34 vinyl groups/nm2 on the surfaces of the 2,4D-CP microspheres prepared via TRPP. The above results revealed that the “living” 2,4-D-MIP/2,4-D-CP microspheres prepared via RAFTPP had much higher densities of surface vinyl groups than the 2,4-D-CP microspheres prepared via TRPP, demonstrating that the “living” mechanism of RAFTPP had significant influence on the properties of the resulting polymer microspheres. On the basis of the above results, it can be concluded that the much higher densities of surface vinyl groups on the “living” 2,4-D-MIP/2,4-D-CP microspheres should be responsible for their obviously higher grafting densities of hydrophilic polymer brushes. Equilibrium Binding Experiments with the Ungrafted and Grafted 2,4-D-MIPs/2,4-D-CPs in Different Media.

18 in Table 1), revealing the presence of rather densely grafted hydrophilic polymer brushes on the MIP/CP particles. It is interesting to note that the molecular weights of the macroCTAs proved to have negligible influence on the grafting densities of hydrophilic polymer brushes in the studied range, which definitely verified the high versatility of this RAFT coupling chemistry. To demonstrate the high efficiency of our strategy for the synthesis of MIP/CP particles with densely grafted hydrophilic polymer brushes, a series of 2,4-D-MIP/2,4-D-CP microspheres with surface-grafted hydrophilic polymer brushes were also prepared via the surface-initiated RAFT polymerization of NIPAAm for different times (t = 12, 24, and 36 h) with the above-obtained “living” 2,4-D-MIP/2,4-D-CP particles with surface-bound dithioester groups (prepared via RAFTPP) as the immobilized RAFT agent in the presence of a certain amount of sacrificial CDB in the polymerization solutions. The weight increases were also observed for these modified MIP and CP particles (entries 19−24 in Table 1), suggesting the successful grafting of PNIPAAm brushes. It is generally accepted that the molecular weights and dispersities of the free polymers generated in the surface-initiated RAFT polymerization systems (due to the addition of sacrificial chain transfer agent) can be utilized to represent those of the grafted polymer brushes.57 Therefore, the free polymers generated in the polymerization solutions were collected and characterized with GPC, from which the number-average molecular weights (Mn,GPC) and molar mass-dispersities (Đ) of the grafted PNIPAAm brushes on the MIP and CP particles were evaluated and summarized in Table 1 (entries 19−24). The grafting densities of the polymer brushes on these grafted MIP/CP microspheres were evaluated to be about 0.9 chains/ nm2 by using their increased weight percentages and abovedescribed method, which are much lower than those of the grafted MIP/CP microspheres prepared via our new strategy. In addition, some grafted polymer microspheres were further prepared by the coupling reaction between a PEG macro-CTA (Mn,PEG = 10000) or PNIPAAm macro-CTA (Mn,NMR = 10300) and the polymer microspheres obtained via TRPP under the identical conditions as RAFTPP but in the absence of the chain transfer agent CDB (these polymer microspheres had only residual vinyl groups on their surfaces, entry 26 in Table 1) (Figure 2b2,b3, entries 27 and 28 in Table 1), which were found to have a grafting density of about 1.0 chain/nm2 by taking their increased weight percentages into consideration. To confirm the accuracy of the grafting densities of the hydrophilic polymer brushes on the grafted MIPs/CPs obtained by the above method, the elemental analyses of some representative ungrafted and grafted 2,4-D-CPs (entries 2, 16, 26, and 28 in Table 1) were also performed to evaluate the grafting densities of the hydrophilic polymer brushes on the grafted 2,4-D-CP particles. The nitrogen contents were found to be 1.21% and 1.91% for the sample entries 2 and 16, respectively, and 1.34% and 2.17% for the sample entries 26 and 28, respectively. By comparing the differences in the nitrogen contents of the ungrafted 2,4-D-CPs (stemming from their incorporated poly(4-VP)) and their corresponding grafted ones (due to both the incorporated poly(4-VP) in the particle cores and the grafted PNIPAAm brushes), the increased weight percentages of the grafted 2,4-D-CP particles (due to the grafting of PNIPAAm brushes) were evaluated to be 6.96% and 8.48% for sample entries 16 and 28, respectively. The grafting densities of PNIPAAm brushes on these grafted 2,4-D-CP 1670


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hydrophilicity.33−35,39,40,43 The molecular weights of the grafted polymer brushes showed considerable influence on the equilibrium binding properties of the MIPs/CPs in pure water. The specific template bindings of the grafted MIPs increased largely in pure water when the chain length of the polymer brushes was increased in the low molecular weight range and then almost leveled off at a molecular weight of ≥2000 for PEG brushes and somewhat larger than 2470 for PNIPAAm brushes, respectively (Figure 4), which demonstrated that only those polymer brushes with high enough molecular weights could act as an efficient hydrophilic protective shield for the MIP particles. In addition, the above results also showed that PEG brushes are somewhat more efficient than PNIPAAm brushes in improving the pure water compatibility of the grafted MIPs (as revealed by the relatively lower molecular weights required for PEG brushes than PNIPAAm brushes for the grafted MIPs to reach their best pure water-compatibility), which might be attributed to the relatively higher hydrophilicity of PEG chains (Table 1). Nevertheless, the grafted MIPs with hydrophilic polymer brushes of different chemical structures and a range of molecular weights could show excellent pure water-compatible template binding properties (i.e., their specific template bindings in pure water were almost the same as those observed in methanol/water (4:1 v/v)) and obvious selectivity toward the template (Figure S8 and Table S3), thus indicating the general applicability of our strategy. Note that a series of 2,4-DMIP/2,4-D-CP microspheres grafted with PNIPAAm brushes of different molecular weights were also prepared via the surface-initiated RAFT polymerization of NIPAAm by using the ungrafted “living” 2,4-D-MIP/2,4-D-CP microspheres as the immobilized RAFT agent (entries 19−24 in Table 1). However, only those grafted MIPs with much longer polymer brushes (Mn ≥ 35300, or grafting time ≥24 h) exhibited good compatibility with pure aqueous solutions in this case (Figure 5), which could be ascribed to their much lower grafting densities of hydrophilic polymer brushes (Table 1). This result in turn demonstrated the high efficiency of RAFT coupling chemistry for the synthesis of water-compatible MIPs. The equilibrium binding experiments were further carried out in the undiluted pure milk and bovine serum. The batch adsorption study revealed that the grafted 2,4-D-MIPs bearing PEG brushes with Mn ≥ 5000 or PNIPAAm brushes with Mn,NMR ≥ 6360 could show obvious specific template bindings in these complex biological media, whereas negligible specific template bindings were observed for those MIPs with shorter PEG or PNIPAAm brushes (Figure 4, Figure S6), which indicated that the chain length of the hydrophilic polymer brushes played a decisive role in the compatibility of the grafted MIPs with biological samples, and only those polymer brushes with a high enough molecular weight could act as an effective hydrophilic protective shield for the MIPs to prevent the accumulation of proteins on their surfaces and enable them to function properly.45−47 It is worth mentioning here that the grafted 2,4-D-MIPs bearing PEG brushes with Mn = 2000 or PNIPAAm brushes with Mn,NMR = 2470 lost their specific template recognition ability in such biological matrices, although they showed obvious specific template bindings in pure aqueous media, demonstrating that it is more difficult to achieve the compatibility of the MIPs with real biological samples (than with pure aqueous solutions) due to their complex nature. Nevertheless, the specific template bindings of the grafted MIPs with different chemical structures and long

The equilibrium binding properties of the ungrafted 2,4-DMIP/2,4-D-CP prepared via RAFTPP and their grafted ones prepared via RAFT coupling chemistry were first studied in an organic solvent-rich medium (i.e., methanol/water (4:1 v/v)). Both the ungrafted and grafted MIPs proved to bind more template than their corresponding CPs (Figure S4) and show high selectivity toward the template (Figure S8 and Table S3), suggesting the presence of specific binding sites in these MIPs. In addition, the specific template bindings (i.e., the binding differences between the MIP and its CP) of the MIP particles in methanol/water were relatively independent of the chemical structures and molecular weights of the grafted polymer brushes (Figure 4), suggesting that the RAFT coupling chemistry had little influence on the binding sites of the MIPs.

Figure 4. Specific template bindings of the ungrafted “living” 2,4-DMIP microspheres prepared via RAFTPP (i.e., Mn of the polymer brush is 0) and their grafted ones with PEG (Mn = 1000, 2000, 5000, and 10000) (a) or PNIPAAm (Mn,NMR = 2470, 6360, 10300, and 14600) (b) brushes in solutions of 2,4-D (0.02 mM) in different media at 25 °C (polymer concentration: 16 mg/mL).

We then performed the equilibrium binding experiments in pure water. As expected, the specific template bindings of the ungrafted 2,4-D-MIP almost completely disappeared in the pure aqueous solution due to their high surface hydrophobicity (Figure 4, Figure S4a).26 In sharp contrast, most of the grafted 2,4-D-MIPs could show obvious specific template bindings in pure aqueous media because of their largely improved surface 1671


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Figure 5. (a) Equilibrium template bindings of the ungrafted “living” 2,4-D-MIP (filled symbols)/2,4-D-CP (open symbols) microspheres (i.e., Mn of the polymer brush = 0 or grafting time t = 0 h) prepared via RAFTPP and their grafted 2,4-D-MIP (filled symbols)/2,4-D-CP (open symbols) microspheres with PNIPAAm brushes (Mn,GPC,MIP = 27600, 35300, and 42000 or grafting time t = 12, 24, and 36 h) prepared via surface-initiated RAFT polymerization in a 2,4-D solution (0.02 mM) in methanol/water (4:1 v/v) (square) and in pure water (triangle) at 25 °C, respectively (polymer concentration: 16 mg/mL). (b) Specific template bindings of the ungrafted “living” 2,4-D-MIP microspheres (i.e., Mn of the polymer brush = 0 or grafting time t = 0) and their grafted ones bearing PNIPAAm brushes (Mn,GPC,MIP = 27600, 35300, and 42000 or grafting time t = 12, 24, and 36 h) prepared via surface-initiated RAFT polymerization in a 2,4-D solution (0.02 mM) in methanol/water (4:1 v/v; filled symbols) and in pure water (open symbols) at 25 °C, respectively (polymer concentration: 16 mg/mL).

Figure 6. Selective bindings of the grafted 2,4-D-MIP/2,4-D-CP microspheres bearing PEG (Mn = 5000 and 10000) or PNIPAAm (Mn,NMR = 6360, 10300, and 14600) brushes prepared via RAFT coupling chemistry toward 2,4-D and POAc in their mixed solution (C2,4‑D or POAc = 0.02 mM) in pure milk (a) and bovine serum (b), respectively (polymer concentration: 16 mg/mL).

enough polymer brushes in pure milk and bovine serum proved to be almost the same as those observed in methanol/water (Figure 4), which clearly confirmed their excellent specific molecular-recognition ability in the real, undiluted biological samples. Figure 6a and b present the selective bindings of the grafted 2,4-D-MIPs/2,4-D-CPs with PEG (Mn = 5000 and 10000) or PNIPAAm (Mn,NMR = 6360, 10300, and 14600) brushes toward 2,4-D and a structurally related compound POAc (which have the same functionality (i.e., carboxyl group) but differ in the numbers of substituents on the benzene ring, Scheme S1) in their mixed solution in pure milk and bovine serum, respectively. It can be seen clearly that although the grafted 2,4-D-MIPs exhibited significantly higher binding capacities toward 2,4-D than toward POAc in real biological solutions, the binding capacities of the grafted 2,4-D-CPs toward 2,4-D were also much higher than those of the grafted 2,4-D-CPs toward POAc, which might be due to their solubility difference in the aqueous solutions.60 The above results suggested that the nonspecific bindings of the studied 2,4-D-MIPs toward 2,4-D

and POAc were quite different, which makes it inappropriate to evaluate the selectivity of the MIPs by directly comparing their binding capacities toward 2,4-D 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.61 IPB can be defined by the following equation: IPB(%) = [(BMIP − BCP)/BCP] × 100

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. The IPB values determined for the studied grafted 2,4-D-MIPs toward 2,4-D and POAc in real biological media are listed in Table S3, which demonstrated clearly that the studied 2,4-D-MIP microspheres grafted with both PNIPAAm and PEG brushes showed obvious specificity toward 2,4-D in the real biological samples. 1672


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General Applicability of the Strategy. A series of propranolol-MIP/propranolol-CP microspheres (propranolol is a sympatholytic nonselective beta blocker) with densely grafted hydrophilic polymer brushes were also prepared by the first synthesis of “living” propranolol-MIP/propranolol-CP microspheres with surface-bound vinyl and dithioester groups via RAFTPP (entries 29 and 30 in Table 1) and their subsequent grafting of hydrophilic polymer brushes through PEG (Mn,PEG = 5000 and 10000) and PNIPAAm (Mn,NMR = 10300 and 14600) macro-CTA-mediated coupling reactions (entries 31−38 in Table 1). Their characterization with SEM (Table 1), FT-IR (Figure S2), and static water contact angle experiments (Table 1) strongly verified the successful synthesis of propranololMIP/propranolol-CP microspheres with surface-grafted hydrophilic polymer brushes. The grafting densities of the hydrophilic polymer brushes on the obtained grafted propranololMIP/propranolol-CP microspheres were determined to be about 1.8 chains/nm2 on the basis of their increased weight percentages after their surface modification, which again demonstrated the high efficiency of the RAFT coupling chemistry. The equilibrium binding properties of the obtained grafted propranolol-MIP microspheres with hydrophilic polymer brushes of different chemical structures and molecular weights were also investigated in different media. The experimental results revealed that they exhibited excellent specific template bindings (Figure 7, Figures S5 and S7) and good selectivity

Figure 8. Selective bindings of the grafted propranolol-MIPs/ propranolol-CPs with PEG (Mn = 5000 and 10000) or PNIPAAm (Mn,NMR = 10300 and 14600) brushes prepared via RAFT coupling chemistry toward propranolol and atenolol in their mixed solution (Cpropranolol or atenolol = 0.1 mM) in pure milk (a) and bovine serum (b), respectively (polymer concentration: 0.6 mg/mL).

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CONCLUSIONS We have demonstrated for the first time the efficient synthesis of a series of well-defined MIP microspheres with densely grafted hydrophilic polymer brushes of desired chemical structures and molecular weights that are compatible with the real, undiluted biological samples by the facile RAFT coupling chemistry. The combined use of RAFTPP and hydrophilic macro-CTA-mediated coupling reaction is central to the successful synthesis of MIP microspheres with high grafting densities of hydrophilic polymer brushes, because RAFTPP has proven to provide polymer particles with much higher densities of surface vinyl groups than those obtained via TRPP. The influencing rule of the structural parameters (including the chemical structures, molecular weights, and grafting densities) of the hydrophilic polymer brushes on the water compatibility of the grafted MIP particles were derived and the general applicability of the strategy was also demonstrated. In view of the facile and controlled synthesis of uniform “living” MIP particles with high densities of surface vinyl groups and adjustable sizes via RAFTPP for a wide range of templates under the normal imprinting conditions, the easy availability of various hydrophilic macro-CTAs either directly via RAFT

Figure 7. Specific template bindings of the ungrafted “living” propranolol-MIP microspheres prepared via RAFTPP (i.e., Mn of the polymer brush is 0) and their grafted ones with PEG (Mn = 5000 and 10000) or PNIPAAm (Mn,NMR = 10300 and 14600) brushes in solutions of propranolol (0.1 mM) in different media at 25 °C (polymer concentration: 0.6 mg/mL).

toward propranolol (Figure 8, Figure S10, Table S5) in both pure water and pure milk and bovine serum. This, together with the total incompatibility of the ungrafted propranolol-MIP with pure water and real biological samples (Figure 7), definitely verified the general applicability and high versatility of our strategy for the preparation of MIP particles with efficient specific recognition of small organic molecules in the complex biological milieu. 1673


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polymerization of hydrophilic monomers or via hydrophilic polymer end-group modification, and the simple implementation of macro-CTA-mediated coupling reaction under mild conditions, we believe the present methodology to represent a general and promising way to develop well-defined real biological sample-compatible MIP particles with great potential as synthetic substitutes for biological receptors in a wide range of analytical applications (e.g., for environmental, food, and clinical analyses) and other areas. We also believe that this work will pave the way for the rational design of water-compatible MIPs, which is of the utmost importance for finally solving this challenging problem in the molecular imprinting field.

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ASSOCIATED CONTENT

S Supporting Information *

Scheme S1, Figures S1−S16, Tables S1−S7, and the detailed equilibrium template binding and competitive binding experiments with the ungrafted and grafted MIPs/CPs in different media. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support from National Natural Science Foundation of China (Grant Nos. 20744003, 20774044, and 21174067), Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20130031110018), and PCSIRT (IRT1257).

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Well-Defined Hydrophilic Molecularly Imprinted Polymer Microspheres

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