Macromolecular Rapid Communications
Communication
Glucose-Sensitive QCM-Sensors Via Direct Surface RAFT Polymerization Caroline Sugnaux, H.-A Klok* Thin, phenylboronic acid-containing polymer coatings are potentially attractive sensory layers for a range of glucose monitoring systems. This contribution presents the synthesis and properties of glucose-sensitive polymer brushes obtained via surface RAFT polymerization of 3-methacrylamido phenylboronic acid (MAPBA). This synthetic strategy is attractive since it allows the controlled growth of PMAPBA brushes with film thicknesses of up to 20 nm via direct polymerization of MAPBA without the need for additional post-polymerization modification or deprotection steps. QCM-D sensor chips modified with a PMAPBA layer respond with a linear change in the shift of the fundamental resonance frequency over a range of physiologically relevant glucose concentrations and are insensitive toward the presence of fructose, thus validating the potential of these polymer brush films as glucose sensory thin coatings.
1. Introduction Boronic acid containing (co)polymers have been attracting increasing interest due to their potential use in a variety of biomedical applications that include the treatment of HIV, obesity, cancer, and diabetes.[1] Treatment of diabetes requires careful, frequent monitoring of blood glucose levels. To this end, numerous polymer-based glucosesensing strategies have been developed, which, in addition to phenyl boronic acid can also be based on the use of glucose oxidase or lectins.[2] While they are very selective toward glucose, glucose oxidase and lectins are potentially susceptible to denaturation, which can be a limitation for long-term use or e.g., when sterilization is required. Boronic acid (co)polymers present a robust and durable glucose-detection platform that has attracted interest to Dr. C. Sugnaux, Prof. H.-A. Klok École Polytechnique Fédérale de Lausanne (EPFL), Institut des Matériaux and Institut des Sciences et Ingénierie Chimiques, Laboratoire des Polymères, Bâtiment MXD, Station 12 CH-1015, Lausanne, Switzerland Fax: + 41 21 693 5650 E-mail: harm-anton.klok@epfl.ch Macromol. Rapid Commun. 2014, DOI: 10.1002/marc.201400217 © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
overcome these drawbacks. The mechanism of the interaction between boronic acids and diols is well-established in the literature.[1,2] Thin polymer coatings containing phenylboronic acid (PBA) residues are attractive platforms for the development of novel glucose sensing systems as they potentially provide faster response times as compared with, e.g., bulk hydrogels. Surface-tethered PBA-containing polymers (“polymer brushes”) have been prepared via free radical polymerization using 3-mercaptopropyl trimethoxysilane modified substrates,[3–5] by grafting dithiolated copolymers obtained via photo-iniferter-mediated or ATRP copolymerization of 3-acrylamidoboronic acid onto gold nanoparticles[6] as well as by post-polymerization modification of poly(meth)acrylic acid brushes obtained via SI-ATRP with 3-amino phenylboronic acid.[7,8] Ivanov et al.[3] and Liu et al.[5] have used PBA-containing polymer brushes to capture cells and subsequently detach them upon exposure of the brush to glucose or fructose. In other examples, PBA-modified polymer brushes were used to study glycoprotein binding[4,6] or for the release of diols.[8] Zauscher and co-workers[7] have grafted PBA-containing polymer brushes from micropatterned gold substrates as well as from microcantilever arrays. These authors demonstrated
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that AFM height images of micropatterned brushes as well as changes in cantilever bending could be used to measure and detect changes in glucose concentration. While conventional free radical polymerization and grafting-onto approaches have also been explored, surface-initiated/mediated controlled radical polymerization techniques are advantageous in that they enable access to high grafting density polymer brushes with well-controlled film thicknesses, composition, and architecture.[9] This contribution presents a new grafting-from strategy for the preparation of PBA-functionalized polymer brushes that obviates the need for post-polymerization modification. Whereas controlled radical polymerization methods, such as ATRP,[10,11] NMP,[12] and RAFT,[13–16] have been extensively used for the direct solution polymerization of a variety of PBA derivatives, these approaches have only found limited use for the preparation of surface-tethered polymer brushes. The strategy presented here is based on the direct surface-RAFT polymerization of 3-methacrylamido phenylboronic acid. In contrast to some of the PBA-containing brushes reported earlier, the approach presented in this contribution allows direct, surface-mediated polymerization of the unprotected monomer, thus circumventing the need for additional post-polymerization modification or deprotection steps. As a first proof-of-concept, the direct surface RAFT polymerization of 3-methacrylamido phenylboronic acid was used to prepare glucose-sensitive QCM sensors. The QCM technique represents an attractive approach that allows to detect mass loading effects and/or changes in viscoelastic properties of surface-attached polymers and has been previously used to develop potassium-selective polymer brush-based sensors.[17] The poly(3-methacrylamido phenylboronic acid) brush-modified QCM sensors presented here were found to display shifts in resonance frequency that were linearly proportional to glucose concentration in the physiologically relevant range. The synthetic strategy reported here may not only be attractive to modify QCM-sensor surfaces but could also be of interest
for other glucose sensing platforms such as gated membrane systems.[18]
2. Experimental Section A detailed description of the materials, analytical methods, as well as thee synthesis of 1 and 2 (Scheme 1) is provided in the Supporting Information.
2.1. Procedures Surface RAFT agent (3) was synthesized as reported previously.[19] Analysis: 1H NMR (CDCl3, 400 MHz), δ (ppm): 7.90 (dd, J = 8.6 and 1.3 Hz, 2H, ArC-H), 7.55 (tt, J = 7.6 and 1.2 Hz, 1H, ArC-H), 7.38 (t, J = 7.7 Hz, 2H, ArC-H), 5.92 (bs, 1H, N-H), 3.83 (q, J = 7.0 Hz, 6H, SiOCH2CH3), 3.25 (q, J = 6.7 and 5.8 Hz, 2H, NHCH2), 2.65–2.35 (m, 4H, CH2CH2C = O), 1.93 (s, 3H, CH3), 1.63 (m, 2H, NHCH2CH2), 1.24 (t, J = 7.0 Hz, 9H, SiOCH2CH3), 0.63 (t, J = 8.0 Hz, 2H, SiCH2CH2CH2NH). (1H NMR spectrum is included in Figure S1 of the Supporting Information).
2.2. Substrate Preparation Silicon wafers and quartz substrates were cut into pieces of 10 mm × 8 mm and 24 mm × 10 mm, respectively. Prior to immobilization of the RAFT agent, the substrates were sonicated for 5 min in ethanol followed by 5 min in acetone, dried under a stream of nitrogen and finally subjected to an oxygen plasma treatment. Silicon oxide quartz crystals (for QCM) were only plasma treated. Oxygen plasma cleaning was performed using a Tepla 300 microwave-induced plasma system (PVA Tepla AG, Germany) during 4 min at 500 W with an oxygen flow rate of 400 mL min−1.
2.3. Immobilization of the Surface RAFT Agent (3).[19] Substrates were immersed under inert atmosphere in a freshly prepared 1 × 10−3 M solution of 3 in dry toluene at room temperature. After 4 h, the modified substrates were rinsed with anhydrous toluene and methylene chloride, three times each, and finally dried under a stream of nitrogen. The surface RAFT agentfunctionalized surfaces were transferred in a reactor and used immediately.
Scheme 1. Preparation of poly(3-methacrylamido phenylboronic acid) (PMAPBA) brushes via surface RAFT polymerization.
2
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2.4. Surface RAFT Polymerization of 3-methacrylamidophenylboronic Acid (1). 4.1 g (20 mmol) of monomer (1) was dissolved in 12.3 mL of a 1:2 (v/v) mixture of water and DMF. The reaction mixture was degassed employing three freeze–pump–thaw cycles and subsequently cannula transferred into a nitrogen-purged reactor containing the surface RAFT agent-modified substrates. After that, 6.5 mg (0.04 mmol) of AIBN ([monomer]:[initiator] = [500]:[1]) was added to the reactor and the polymerization was carried out at 70 °C for the desired reaction time. After polymerization, the substrates were taken from the reaction mixture and extensively washed with DMF and ethanol and finally dried under a flow of nitrogen.
2.5. Surface RAFT Polymerization of N-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methacrylamide (2). 4.0 g (13.88 mmol) of monomer (2) was dissolved in 7.2 mL of DMF. The reaction mixture was degassed employing three freeze–pump–thaw cycles and subsequently cannula transferred into a nitrogen-purged reactor containing the surface RAFT agent-modified substrates. After that, 4.5 mg (0.027 mmol) of AIBN ([monomer]:[initiator] = [500]:[1]) was added to the reactor and the polymerization was carried out at 70 °C for the desired reaction time. After polymerization, the substrates were taken from the reaction mixture and extensively washed with DMF and ethanol and finally dried under a flow of nitrogen.
3. Results and Discussion The synthesis of poly(3-methacrylamido phenylboronic acid) (PMAPBA) brushes via the SI-RAFT process starts with the immobilization of surface RAFT agent 3, which promotes RAFT polymerization via the R-group approach,[19] on silicon oxide surfaces as illustrated in Scheme 1. Surface immobilization of 3 resulted in an increase of water contact angle from 0° to 66° confirming the modification of the silicon substrate with the RAFT agent. The surface RAFT polymerization of 1 was then carried out at 70 °C using AIBN as the initiator at a [monomer] : [initiator] ratio of 500 : 1. Film thicknesses of the polymer brushes were determined using AFM analysis on micropatterned samples, which were obtained from substrates modified with 3 that were patterned via a previously reported process (see also Figure S4A, Supporting Information).[19,20] As shown in Figure 1, the surface RAFT strategy outlined in Scheme 1 allows access to PMAPBA brushes with film thicknesses of up to ≈22 nm. In addition to boronic acid 1, the RAFT agentmodified substrates also enabled surface polymerization of its protected analogue N-(3-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)phenyl)methacrylamide (TMDPMA) (2). After a reaction time of 120 min, a PTMDPMA brush with a thickness of 17 nm was obtained (Figure S4B, Supporting
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Figure 1. Evolution of the film thickness of poly(3-methacrylamido phenylboronic acid) (PMAPBA) brushes as a function of polymerization time.
Information). While the approach outlined in Scheme 1 enables direct surface RAFT polymerization of both 2 and 1, all of the analyses and characterization experiments that will be discussed in the remainder of this paper have been performed on brushes obtained via (direct) polymerization of 1 as this avoids an additional deprotection step. The PMAPBA brushes were characterized with FTIR and UV–vis spectroscopy as well as XPS. Figure S5A (Supporting Information) shows the FTIR spectrum of a 19-nm thick PMAPBA brush. The broad absorbance band at ≈3400 cm−1 corresponds to the OH stretching of the boronic acid group. Asymmetric and symmetric CH2-stretching vibrations are present at ≈2920 and ≈2850 cm−1, respectively, as well as a strong C = O stretch vibration band at ≈1645 cm−1, which is due to the amide group. At lower energy, N–H bending and C–N stretching vibrations absorb at ≈1538 cm−1, confirming the presence of monosubstituted amides. Finally, the absorption at ≈1344 cm−1 is attributed to the B-O bond stretching. Figure S5B (Supporting Information) shows the UV–vis spectrum of a 12-nm thick PMAPBA brush, which reveals two absorption peaks at 212 and 244 nm, reflecting the presence of the phenylboronic acid groups. Figure 2 shows an XPS survey scan as well as C1s and O1s high resolution scans recorded from a 19-nm thick PMAPBA brush. In agreement with the chemical composition of the polymer brush, the survey scan reveals O, N, C, and B signals. The N1s signal at 400.2 eV is due to the amide bond, while the B1s signal at 191.6 eV illustrates the presence of the boronic acid group.[21] The C1s corelevel spectrum can be curve-fitted with four components with the expected relative ratios, which appear at 284.5, 285.0, 285.7, and 288.0 eV, respectively, and can be attributed to the aromatic (C-ring) and aliphatic carbon atoms
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Figure 2. A) Chemical structure of a PMAPBA brush. B) Survey, C) C 1s core level, and D) O 1s core level XPS scans of a 19-nm thick PMAPBA brush (the numbers and symbols in the core-level spectra refer to the respective atoms in the chemical structure).
(C–H), substituted aromatic carbons and quaternary carbon, and carbonyl (C = O) carbon atoms of the polymer brush. The O1s high-resolution scan can be deconvoluted in two components at 531.3 and 532.7 eV with relative intensities of 1:2, which are due to the amide, respectively, boronic acid oxygen atoms. The corresponding survey and high-resolution scans of a brush prepared from 2 are included in Figure S6 (Supporting Information). The glucose sensitivity of the PMAPBA brushes was assessed using QCM-D experiments. To this end, a QCM-D chip modified with a 12-nm thick PMAPBA brush was first stabilized using a 10 × 10−3 M PBS buffer at pH 9 and subsequently exposed to 10 × 10−3 M PBS solutions with increasing glucose concentrations (at pH 9). These experiments were carried out at pH 9 due to the pKa of the phenyl boronic acid moiety, which is ≈8.8.[22] Figure 3A illustrates the changes in the shift of the fundamental resonance frequency (Δf) and the dissipation (D) upon exposing the PMAPBA brush to glucose concentrations up to 100 × 10−3M. With increasing glucose concentration, the shift in the fundamental resonance frequency becomes more negative, while simultaneously a relatively small increase in the dissipation signal is observed. The PMAPBA-modified QCM-D sensor responds with a relatively linear change in Δf over a range of glucose concentrations varying from 5 to 50 × 10−3M (Figure 3B; the gray area highlights the physiologically relevant, normal, and
4
hyperglycemic range of blood glucose concentrations). Figure 3C illustrates the changes in the fundamental harmonic resonance upon repetitive exposure of a 12-nm thick PMAPBA coated QCM-D sensor to pH 9 PBS buffer solutions with and without 25 × 10−3M glucose. Over a sequence of seven repetitive cycles, the QCM-D-based sensor showed a complete reversible and reproducible response. While glucose is predominant (6 × 10−3 M for healthy adults), human blood also contains other sugars such as fructose. Although present at much lower concentrations (fructose concentrations in human blood vary from 8.1 ± 1.0 × 10−6 M for healthy to 12.0 ± 3.8 × 10−6 M diabetic adults),[23] fructose has been reported to have a much higher binding constant toward PBA as compared with glucose (560 M−1, respectively, 11 M−1 at pH 8.5 in 0.1 M phosphate buffer).[24] To assess the influence of possible competitive binding of fructose on the glucose response of the PMAPBA-modified QCM-D chips, an experiment was carried out in which the sensor chip was exposed to 5 × 10−3 M glucose in 10 × 10−3 M PBS with and without 10 × 10−6 M fructose. As illustrated by the results shown in Figure 3D, the shift in the fundamental harmonic response was not influenced by the presence of 10 × 10−6 M fructose, indicating that the QCM-based sensor is able to detect physiologically relevant glucose concentrations[25] also in the presence of other competing sugars. The data
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Figure 3. A) Shifts of the first harmonic frequency and the dissipation signal recorded for a QCM-D chip modified with a 12-nm thick PMAPBA brush upon exposure to pH 9 PBS buffer solutions containing up to 100 × 10−3 M glucose; B) Shift in the fundamental harmonic resonance of a 12-nm thick PMAPBA brush coated silicon QCM chip as a function of glucose concentration (error bars represent the standard deviation over three independent measurements on the same QCM chip). The shaded area indicates the physiologically relevant range of glucose concentrations[25]; C) Fundamental harmonic resonance response of a 12-nm thick PMAPBA brush coated QCM chip, which was exposed alternatingly to pH 9 PBS solutions with or without 25 × 10−3 M glucose over seven cycles; D) Shift in the fundamental harmonic resonance response of a 12-nm thick PMAPBA brush coated QCM chip, which was exposed to pH 9 PBS solutions containing 5 × 10−3 M glucose with or without 10 × 10−6 M fructose.
in Figure 3 are from experiments on a single-PMAPBAmodified QCM-D chip, which was selected as a representative example. QCM-D sensograms representing the shifts of the first harmonic frequency and the dissipation signal upon exposure to pH 9 PBS buffer solutions containing up to 100 × 10−3 M glucose for two other PMAPBA-modified QCM-D chips are included in the Supporting Information (Figure S7).
4. Conclusions This manuscript has presented a novel strategy for the preparation of thin PBA-containing polymer brush films, which is based on the surface-RAFT polymerization of
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3-methacrylamido phenylboronic acid (MAPBA). This strategy, which allows the controlled growth of PMAPBA brushes with thicknesses up to ≈20 nm, is attractive as it circumvents the need for additional post-polymerization modification and deprotection steps. The surface RAFT polymerization of MAPBA was used to modify QCM-D sensor chips. Upon exposure to pH 9 PBS solutions of glucose, PMAPBA brush-modified QCM-D chips showed shifts in the fundamental resonance frequency that varied linearly over a range of physiologically relevant glucose concentrations and which were not disturbed by competitive binding of fructose. While the relatively high pKa value of the PBA moiety (≈8.8) will not allow in vivo glucose monitoring or the direct analysis of serum samples, the PBA-containing polymer brush coatings can be apply to
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study samples that are diluted in an appropriate (pH 9) buffer. The synthetic strategy presented herein may not only be useful to generate glucose-sensitive QCM-D sensors but could also be an attractive method to prepare glucose-responsive thin films for a variety of other sensor technologies.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author. Acknowledgements: This research was partially supported through FP7 grant number 247138 from the European commission to the AP@home consortium (www.apathome. eu) as well as via the European Science Foundation Precision Polymer Materials (P2M) Research Networking Programme. Received: April 13, 2014; Revised: May Published online: ; DOI: 10.1002/marc.201400217
21,
2014;
Keywords: boronic acid; glucose; polymer brush; quartz crystal microbalance; sensor [1] J. N. Cambre, B. S. Sumerlin, Polymer 2011, 52, 4631. [2] Q. Wu, L. Wang, H. Yu, J. Wang, Z. Chen, Chem. Rev. 2011, 111, 7855. [3] A. E. Ivanov, J. Eccles, H. A. Panahi, A. Kumar, M. V. Kuzimenkova, L. Nilsson, B. Bergenståhl, N. Long, G. J. Phillips, S. V. Mikhalovsky, I. Y. Galaev, B. Mattiasson, J. Biomed. Mater. Res., Part A 2009, 88A, 213. [4] A. E. Ivanov, N. Solodukhina, M. Wahlgren, L. Nilsson, A. A. Vikhrov, M. P. Nikitin, A. V. Orlov, P. I. Nikitin, M. V. Kuzimenkova, V. P. Zubov, Macromol. Biosci. 2011, 11, 275.
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[5] H. Liu, Y. Li, K. Sun, J. Fan, P. Zhang, J. Meng, S. Wang, L. Jiang, J. Am. Chem. Soc. 2013, 135, 7603. [6] Y. Anraku, Y. Takahashi, H. Kitano, M. Hakari, Colloids Surf., B 2007, 57, 61. [7] T. Chen, D. P. Chang, T. Liu, R. Desikan, R. Datar, T. Thundat, R. Berger, S. Zauscher, J. Mater. Chem. 2010, 20, 3391. [8] H.-B. Liu, Q. Yan, C. Wang, X. Liu, C. Wang, X.-H. Zhou, S.-J. Xiao, Colloids Surf., A 2011, 386, 131. [9] R. Barbey, L. Lavanant, D. Paripovic, N. Schüwer, C. Sugnaux, S. Tugulu, H.-A. Klok, Chem. Rev. 2009, 109, 5437. [10] Y. Qin, V. Sukul, D. Pagakos, C. Cui, F. Jäkle, Macromolecules 2005, 38, 8987. [11] Y. Yao, X. Wang, T. Tan, J. Yang, Soft Matter 2011, 7, 7948. [12] G. Vancoillie, S. Pelz, E. Holder, R. Hoogenboom, Polym. Chem. 2012, 3, 1726. [13] J. N. Cambre, D. Roy, S. R. Gondi, B. S. Sumerlin, J. Am. Chem. Soc. 2007, 129, 10348. [14] K. T. Kim, J. J. L. M. Cornelissen, R. J. M. Nolte, J. C. M. van Hest, J. Am. Chem. Soc. 2009, 131, 13908. [15] D. Roy, J. N. Cambre, B. S. Sumerlin, Chem. Commun. 2008, 2477. [16] S. Maji, G. Vancoillie, L. Voorhaar, Q. Zhang, R. Hoogenboom, Macromol. Rapid Commun. 2014, 35, 214. [17] N. Schüwer, H.-A. Klok, Adv. Mater. 2010, 22, 3251. [18] C. Sugnaux, L. Lavanant, H.-A. Klok, Langmuir 2013, 29, 7325. [19] K. A. Günay, N. Schüwer, H.-A. Klok, Polym. Chem. 2012, 3, 2186. [20] S. Tugulu, M. Harms, M. Fricke, D. Volkmer, H.-A. Klok, Angew. Chem Int. Ed. 2006, 45, 7458. [21] W. Wu, H. Zhu, L. Fan, D. Liu, R. Renneberg, S. Yang, Chem. Commun. 2007, 2345. [22] G. Springsteen, B. Wang, Tetrahedron 2002, 58, 5291. [23] T. Kawasaki, H. Akanuma, T. Yamanouchi, Diabetes Care 2002, 25, 353. [24] J. Yan, G. Springsteen, S. Deeter, B. Wang, Tetrahedron 2004, 60, 11205. [25] Diabetes Care 2012, 35, S11.
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Communication
Glucose-Sensitive QCM-Sensors Via Direct Surface RAFT Polymerization Caroline Sugnaux, H.-A Klok* Thin, phenylboronic acid-containing polymer coatings are potentially attractive sensory layers for a range of glucose monitoring systems. This contribution presents the synthesis and properties of glucose-sensitive polymer brushes obtained via surface RAFT polymerization of 3-methacrylamido phenylboronic acid (MAPBA). This synthetic strategy is attractive since it allows the controlled growth of PMAPBA brushes with film thicknesses of up to 20 nm via direct polymerization of MAPBA without the need for additional post-polymerization modification or deprotection steps. QCM-D sensor chips modified with a PMAPBA layer respond with a linear change in the shift of the fundamental resonance frequency over a range of physiologically relevant glucose concentrations and are insensitive toward the presence of fructose, thus validating the potential of these polymer brush films as glucose sensory thin coatings.
1. Introduction Boronic acid containing (co)polymers have been attracting increasing interest due to their potential use in a variety of biomedical applications that include the treatment of HIV, obesity, cancer, and diabetes.[1] Treatment of diabetes requires careful, frequent monitoring of blood glucose levels. To this end, numerous polymer-based glucosesensing strategies have been developed, which, in addition to phenyl boronic acid can also be based on the use of glucose oxidase or lectins.[2] While they are very selective toward glucose, glucose oxidase and lectins are potentially susceptible to denaturation, which can be a limitation for long-term use or e.g., when sterilization is required. Boronic acid (co)polymers present a robust and durable glucose-detection platform that has attracted interest to Dr. C. Sugnaux, Prof. H.-A. Klok École Polytechnique Fédérale de Lausanne (EPFL), Institut des Matériaux and Institut des Sciences et Ingénierie Chimiques, Laboratoire des Polymères, Bâtiment MXD, Station 12 CH-1015, Lausanne, Switzerland Fax: + 41 21 693 5650 E-mail: harm-anton.klok@epfl.ch Macromol. Rapid Commun. 2014, DOI: 10.1002/marc.201400217 © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
overcome these drawbacks. The mechanism of the interaction between boronic acids and diols is well-established in the literature.[1,2] Thin polymer coatings containing phenylboronic acid (PBA) residues are attractive platforms for the development of novel glucose sensing systems as they potentially provide faster response times as compared with, e.g., bulk hydrogels. Surface-tethered PBA-containing polymers (“polymer brushes”) have been prepared via free radical polymerization using 3-mercaptopropyl trimethoxysilane modified substrates,[3–5] by grafting dithiolated copolymers obtained via photo-iniferter-mediated or ATRP copolymerization of 3-acrylamidoboronic acid onto gold nanoparticles[6] as well as by post-polymerization modification of poly(meth)acrylic acid brushes obtained via SI-ATRP with 3-amino phenylboronic acid.[7,8] Ivanov et al.[3] and Liu et al.[5] have used PBA-containing polymer brushes to capture cells and subsequently detach them upon exposure of the brush to glucose or fructose. In other examples, PBA-modified polymer brushes were used to study glycoprotein binding[4,6] or for the release of diols.[8] Zauscher and co-workers[7] have grafted PBA-containing polymer brushes from micropatterned gold substrates as well as from microcantilever arrays. These authors demonstrated
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that AFM height images of micropatterned brushes as well as changes in cantilever bending could be used to measure and detect changes in glucose concentration. While conventional free radical polymerization and grafting-onto approaches have also been explored, surface-initiated/mediated controlled radical polymerization techniques are advantageous in that they enable access to high grafting density polymer brushes with well-controlled film thicknesses, composition, and architecture.[9] This contribution presents a new grafting-from strategy for the preparation of PBA-functionalized polymer brushes that obviates the need for post-polymerization modification. Whereas controlled radical polymerization methods, such as ATRP,[10,11] NMP,[12] and RAFT,[13–16] have been extensively used for the direct solution polymerization of a variety of PBA derivatives, these approaches have only found limited use for the preparation of surface-tethered polymer brushes. The strategy presented here is based on the direct surface-RAFT polymerization of 3-methacrylamido phenylboronic acid. In contrast to some of the PBA-containing brushes reported earlier, the approach presented in this contribution allows direct, surface-mediated polymerization of the unprotected monomer, thus circumventing the need for additional post-polymerization modification or deprotection steps. As a first proof-of-concept, the direct surface RAFT polymerization of 3-methacrylamido phenylboronic acid was used to prepare glucose-sensitive QCM sensors. The QCM technique represents an attractive approach that allows to detect mass loading effects and/or changes in viscoelastic properties of surface-attached polymers and has been previously used to develop potassium-selective polymer brush-based sensors.[17] The poly(3-methacrylamido phenylboronic acid) brush-modified QCM sensors presented here were found to display shifts in resonance frequency that were linearly proportional to glucose concentration in the physiologically relevant range. The synthetic strategy reported here may not only be attractive to modify QCM-sensor surfaces but could also be of interest
for other glucose sensing platforms such as gated membrane systems.[18]
2. Experimental Section A detailed description of the materials, analytical methods, as well as thee synthesis of 1 and 2 (Scheme 1) is provided in the Supporting Information.
2.1. Procedures Surface RAFT agent (3) was synthesized as reported previously.[19] Analysis: 1H NMR (CDCl3, 400 MHz), δ (ppm): 7.90 (dd, J = 8.6 and 1.3 Hz, 2H, ArC-H), 7.55 (tt, J = 7.6 and 1.2 Hz, 1H, ArC-H), 7.38 (t, J = 7.7 Hz, 2H, ArC-H), 5.92 (bs, 1H, N-H), 3.83 (q, J = 7.0 Hz, 6H, SiOCH2CH3), 3.25 (q, J = 6.7 and 5.8 Hz, 2H, NHCH2), 2.65–2.35 (m, 4H, CH2CH2C = O), 1.93 (s, 3H, CH3), 1.63 (m, 2H, NHCH2CH2), 1.24 (t, J = 7.0 Hz, 9H, SiOCH2CH3), 0.63 (t, J = 8.0 Hz, 2H, SiCH2CH2CH2NH). (1H NMR spectrum is included in Figure S1 of the Supporting Information).
2.2. Substrate Preparation Silicon wafers and quartz substrates were cut into pieces of 10 mm × 8 mm and 24 mm × 10 mm, respectively. Prior to immobilization of the RAFT agent, the substrates were sonicated for 5 min in ethanol followed by 5 min in acetone, dried under a stream of nitrogen and finally subjected to an oxygen plasma treatment. Silicon oxide quartz crystals (for QCM) were only plasma treated. Oxygen plasma cleaning was performed using a Tepla 300 microwave-induced plasma system (PVA Tepla AG, Germany) during 4 min at 500 W with an oxygen flow rate of 400 mL min−1.
2.3. Immobilization of the Surface RAFT Agent (3).[19] Substrates were immersed under inert atmosphere in a freshly prepared 1 × 10−3 M solution of 3 in dry toluene at room temperature. After 4 h, the modified substrates were rinsed with anhydrous toluene and methylene chloride, three times each, and finally dried under a stream of nitrogen. The surface RAFT agentfunctionalized surfaces were transferred in a reactor and used immediately.
Scheme 1. Preparation of poly(3-methacrylamido phenylboronic acid) (PMAPBA) brushes via surface RAFT polymerization.
2
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Glucose-Sensitive QCM-Sensors Via Direct Surface RAFT Polymerization
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2.4. Surface RAFT Polymerization of 3-methacrylamidophenylboronic Acid (1). 4.1 g (20 mmol) of monomer (1) was dissolved in 12.3 mL of a 1:2 (v/v) mixture of water and DMF. The reaction mixture was degassed employing three freeze–pump–thaw cycles and subsequently cannula transferred into a nitrogen-purged reactor containing the surface RAFT agent-modified substrates. After that, 6.5 mg (0.04 mmol) of AIBN ([monomer]:[initiator] = [500]:[1]) was added to the reactor and the polymerization was carried out at 70 °C for the desired reaction time. After polymerization, the substrates were taken from the reaction mixture and extensively washed with DMF and ethanol and finally dried under a flow of nitrogen.
2.5. Surface RAFT Polymerization of N-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methacrylamide (2). 4.0 g (13.88 mmol) of monomer (2) was dissolved in 7.2 mL of DMF. The reaction mixture was degassed employing three freeze–pump–thaw cycles and subsequently cannula transferred into a nitrogen-purged reactor containing the surface RAFT agent-modified substrates. After that, 4.5 mg (0.027 mmol) of AIBN ([monomer]:[initiator] = [500]:[1]) was added to the reactor and the polymerization was carried out at 70 °C for the desired reaction time. After polymerization, the substrates were taken from the reaction mixture and extensively washed with DMF and ethanol and finally dried under a flow of nitrogen.
3. Results and Discussion The synthesis of poly(3-methacrylamido phenylboronic acid) (PMAPBA) brushes via the SI-RAFT process starts with the immobilization of surface RAFT agent 3, which promotes RAFT polymerization via the R-group approach,[19] on silicon oxide surfaces as illustrated in Scheme 1. Surface immobilization of 3 resulted in an increase of water contact angle from 0° to 66° confirming the modification of the silicon substrate with the RAFT agent. The surface RAFT polymerization of 1 was then carried out at 70 °C using AIBN as the initiator at a [monomer] : [initiator] ratio of 500 : 1. Film thicknesses of the polymer brushes were determined using AFM analysis on micropatterned samples, which were obtained from substrates modified with 3 that were patterned via a previously reported process (see also Figure S4A, Supporting Information).[19,20] As shown in Figure 1, the surface RAFT strategy outlined in Scheme 1 allows access to PMAPBA brushes with film thicknesses of up to ≈22 nm. In addition to boronic acid 1, the RAFT agentmodified substrates also enabled surface polymerization of its protected analogue N-(3-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)phenyl)methacrylamide (TMDPMA) (2). After a reaction time of 120 min, a PTMDPMA brush with a thickness of 17 nm was obtained (Figure S4B, Supporting
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Figure 1. Evolution of the film thickness of poly(3-methacrylamido phenylboronic acid) (PMAPBA) brushes as a function of polymerization time.
Information). While the approach outlined in Scheme 1 enables direct surface RAFT polymerization of both 2 and 1, all of the analyses and characterization experiments that will be discussed in the remainder of this paper have been performed on brushes obtained via (direct) polymerization of 1 as this avoids an additional deprotection step. The PMAPBA brushes were characterized with FTIR and UV–vis spectroscopy as well as XPS. Figure S5A (Supporting Information) shows the FTIR spectrum of a 19-nm thick PMAPBA brush. The broad absorbance band at ≈3400 cm−1 corresponds to the OH stretching of the boronic acid group. Asymmetric and symmetric CH2-stretching vibrations are present at ≈2920 and ≈2850 cm−1, respectively, as well as a strong C = O stretch vibration band at ≈1645 cm−1, which is due to the amide group. At lower energy, N–H bending and C–N stretching vibrations absorb at ≈1538 cm−1, confirming the presence of monosubstituted amides. Finally, the absorption at ≈1344 cm−1 is attributed to the B-O bond stretching. Figure S5B (Supporting Information) shows the UV–vis spectrum of a 12-nm thick PMAPBA brush, which reveals two absorption peaks at 212 and 244 nm, reflecting the presence of the phenylboronic acid groups. Figure 2 shows an XPS survey scan as well as C1s and O1s high resolution scans recorded from a 19-nm thick PMAPBA brush. In agreement with the chemical composition of the polymer brush, the survey scan reveals O, N, C, and B signals. The N1s signal at 400.2 eV is due to the amide bond, while the B1s signal at 191.6 eV illustrates the presence of the boronic acid group.[21] The C1s corelevel spectrum can be curve-fitted with four components with the expected relative ratios, which appear at 284.5, 285.0, 285.7, and 288.0 eV, respectively, and can be attributed to the aromatic (C-ring) and aliphatic carbon atoms
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Figure 2. A) Chemical structure of a PMAPBA brush. B) Survey, C) C 1s core level, and D) O 1s core level XPS scans of a 19-nm thick PMAPBA brush (the numbers and symbols in the core-level spectra refer to the respective atoms in the chemical structure).
(C–H), substituted aromatic carbons and quaternary carbon, and carbonyl (C = O) carbon atoms of the polymer brush. The O1s high-resolution scan can be deconvoluted in two components at 531.3 and 532.7 eV with relative intensities of 1:2, which are due to the amide, respectively, boronic acid oxygen atoms. The corresponding survey and high-resolution scans of a brush prepared from 2 are included in Figure S6 (Supporting Information). The glucose sensitivity of the PMAPBA brushes was assessed using QCM-D experiments. To this end, a QCM-D chip modified with a 12-nm thick PMAPBA brush was first stabilized using a 10 × 10−3 M PBS buffer at pH 9 and subsequently exposed to 10 × 10−3 M PBS solutions with increasing glucose concentrations (at pH 9). These experiments were carried out at pH 9 due to the pKa of the phenyl boronic acid moiety, which is ≈8.8.[22] Figure 3A illustrates the changes in the shift of the fundamental resonance frequency (Δf) and the dissipation (D) upon exposing the PMAPBA brush to glucose concentrations up to 100 × 10−3M. With increasing glucose concentration, the shift in the fundamental resonance frequency becomes more negative, while simultaneously a relatively small increase in the dissipation signal is observed. The PMAPBA-modified QCM-D sensor responds with a relatively linear change in Δf over a range of glucose concentrations varying from 5 to 50 × 10−3M (Figure 3B; the gray area highlights the physiologically relevant, normal, and
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hyperglycemic range of blood glucose concentrations). Figure 3C illustrates the changes in the fundamental harmonic resonance upon repetitive exposure of a 12-nm thick PMAPBA coated QCM-D sensor to pH 9 PBS buffer solutions with and without 25 × 10−3M glucose. Over a sequence of seven repetitive cycles, the QCM-D-based sensor showed a complete reversible and reproducible response. While glucose is predominant (6 × 10−3 M for healthy adults), human blood also contains other sugars such as fructose. Although present at much lower concentrations (fructose concentrations in human blood vary from 8.1 ± 1.0 × 10−6 M for healthy to 12.0 ± 3.8 × 10−6 M diabetic adults),[23] fructose has been reported to have a much higher binding constant toward PBA as compared with glucose (560 M−1, respectively, 11 M−1 at pH 8.5 in 0.1 M phosphate buffer).[24] To assess the influence of possible competitive binding of fructose on the glucose response of the PMAPBA-modified QCM-D chips, an experiment was carried out in which the sensor chip was exposed to 5 × 10−3 M glucose in 10 × 10−3 M PBS with and without 10 × 10−6 M fructose. As illustrated by the results shown in Figure 3D, the shift in the fundamental harmonic response was not influenced by the presence of 10 × 10−6 M fructose, indicating that the QCM-based sensor is able to detect physiologically relevant glucose concentrations[25] also in the presence of other competing sugars. The data
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Glucose-Sensitive QCM-Sensors Via Direct Surface RAFT Polymerization
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Figure 3. A) Shifts of the first harmonic frequency and the dissipation signal recorded for a QCM-D chip modified with a 12-nm thick PMAPBA brush upon exposure to pH 9 PBS buffer solutions containing up to 100 × 10−3 M glucose; B) Shift in the fundamental harmonic resonance of a 12-nm thick PMAPBA brush coated silicon QCM chip as a function of glucose concentration (error bars represent the standard deviation over three independent measurements on the same QCM chip). The shaded area indicates the physiologically relevant range of glucose concentrations[25]; C) Fundamental harmonic resonance response of a 12-nm thick PMAPBA brush coated QCM chip, which was exposed alternatingly to pH 9 PBS solutions with or without 25 × 10−3 M glucose over seven cycles; D) Shift in the fundamental harmonic resonance response of a 12-nm thick PMAPBA brush coated QCM chip, which was exposed to pH 9 PBS solutions containing 5 × 10−3 M glucose with or without 10 × 10−6 M fructose.
in Figure 3 are from experiments on a single-PMAPBAmodified QCM-D chip, which was selected as a representative example. QCM-D sensograms representing the shifts of the first harmonic frequency and the dissipation signal upon exposure to pH 9 PBS buffer solutions containing up to 100 × 10−3 M glucose for two other PMAPBA-modified QCM-D chips are included in the Supporting Information (Figure S7).
4. Conclusions This manuscript has presented a novel strategy for the preparation of thin PBA-containing polymer brush films, which is based on the surface-RAFT polymerization of
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3-methacrylamido phenylboronic acid (MAPBA). This strategy, which allows the controlled growth of PMAPBA brushes with thicknesses up to ≈20 nm, is attractive as it circumvents the need for additional post-polymerization modification and deprotection steps. The surface RAFT polymerization of MAPBA was used to modify QCM-D sensor chips. Upon exposure to pH 9 PBS solutions of glucose, PMAPBA brush-modified QCM-D chips showed shifts in the fundamental resonance frequency that varied linearly over a range of physiologically relevant glucose concentrations and which were not disturbed by competitive binding of fructose. While the relatively high pKa value of the PBA moiety (≈8.8) will not allow in vivo glucose monitoring or the direct analysis of serum samples, the PBA-containing polymer brush coatings can be apply to
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study samples that are diluted in an appropriate (pH 9) buffer. The synthetic strategy presented herein may not only be useful to generate glucose-sensitive QCM-D sensors but could also be an attractive method to prepare glucose-responsive thin films for a variety of other sensor technologies.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author. Acknowledgements: This research was partially supported through FP7 grant number 247138 from the European commission to the AP@home consortium (www.apathome. eu) as well as via the European Science Foundation Precision Polymer Materials (P2M) Research Networking Programme. Received: April 13, 2014; Revised: May Published online: ; DOI: 10.1002/marc.201400217
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2014;
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