Mechanism of Surface Molecular Imprinting in Polyelectrolyte

Electrostatics and Charge Regulation in Polyelectrolyte Multilayered Assembly. Andrey G. Cherstvy ... Correlating the Compliance and Permeability of P...
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Mechanism of Surface Molecular Imprinting in Polyelectrolyte Multilayers Jan Gauczinski,† Zan Liu,‡ Xi Zhang,‡ and Monika Sch€onhoff*,† †

Westf€ alische Wilhelms Universit€ at, Institute of Physical Chemistry, Corrensstrasse 28/30, 48149 M€ unster, Germany, and ‡Key Laboratory of Organic Optoelectronics & Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China Received January 18, 2010. Revised Manuscript Received March 15, 2010

The combination of Layer-by-Layer (LbL) self-assembly of oppositely charged polymers with the concept of molecular imprinting in polymers promises faster loading/unloading as compared to bulk systems. Here, we monitor the construction of LbL self-assembled polyelectrolyte multilayers (PEM) including template molecules and the binding and release dynamics of the guest molecules in the imprinted sites employing a quartz crystal microbalance with dissipation measurement (QCM-D). It is found that pH-dependent removal and rebinding of the template leads to a simultaneous swelling of the film. Separating the swelling from the template kinetics is a task which can be carried out by careful interpretation of the obtained QCM-D data. Considering correlated frequency and dissipation changes, evidence is found that the film features binding sites that can be loaded with the template such that the major part of template uptake is due to selective binding into imprinted sites. Template uptake is causing an enhanced cross-linking, as monitored by a reduced dissipation. The mechanism of reversible template uptake and release is shown to be based on the charge equilibrium in the film, which is manipulated by pH variation.

Introduction The formation of molecularly imprinted polymers (MIPs), mimicking complex molecular recognition systems in nature, is a promising method to obtain systems for specific separation or reactive sites for nanoreactors.1-3 Research on MIPs has increased rapidly in recent years and can find application in many different fields, such as separation,1,4 sensors for chemical or biochemical active components,5-7 catalysis,8-11 or novel drug design12 for its easy preparation and thermal and chemical stability, and highly selective recognition capabilities. However, the MIPs suffer some intrinsic limitations from their preparation procedure including the deeply buried binding sites in bulk hindering the subsequent template removal and guest molecule diffusion process, thereby decreasing the imprinting efficiency. Even improvements such as the addition of porogen (pore generating reagent) and the crush of the bulk material have disadvantages, as they bring impurities to the imprinting system or cause damage of the binding sites. A possible solution to these drawbacks is to establish novel molecular recognition systems within the dimension of nanoscales to allow a large fraction of the *Corresponding author. E-mail: [email protected], Phone: þ492518323419, Fax þ49-2518329138.

(1) Wulff, G. Angew. Chem., Int. Ed. Engl. 1995, 34, 1812. (2) Haupt, K.; Mosbach, K. Chem. Rev. 2000, 100, 2495. (3) Alexander, C.; Andersson, H. S.; Andersson, L. I.; Ansell, R. J.; Kirsch, N.; Nicholls, I. A.; O’Mahony, J.; Whitcombe, M. J. J. Mol. Recognit. 2006, 19, 106. (4) Wang, J. F.; Cormack, P. A. G.; Sherrington, D. C.; Khoshdel, E. Angew. Chem., Int. Ed. Engl. 2003, 42, 5336. (5) Liang, C. D.; Peng, H.; Bao, X. Y.; Nie, L. H.; Yao, S. Z. Analyst 1999, 124, 1781. (6) Kroger, S.; Turner, A. P. F.; Mosbach, K.; Haupt, K. Anal. Chem. 1999, 71, 3698. (7) Kriz, D.; Ramstrom, O.; Svensson, A.; Mosbach, K. Anal. Chem. 1995, 67, 2142. (8) Strikovsky, A. G.; Kasper, D.; Grun, M.; Green, B. S.; Hradil, J.; Wulff, G. J. Am. Chem. Soc. 2000, 122, 6295. (9) Robinson, D. K.; Mosbach, K. J. Chem. Soc., Chem. Commun. 1989, 969. (10) Polborn, K.; Severin, K. Chem. Commun. 1999, 2481. (11) Liu, J. Q.; Wulff, G. Angew. Chem., Int. Ed. Engl. 2004, 43, 1287. (12) Allender, C. J.; Richardson, C.; Woodhouse, B.; Heard, C. M.; Brain, K. R. Int. J. Pharm. 2000, 195, 39.

10122 DOI: 10.1021/la1002447

binding sites to be located at or near the surface. Attempts have been made to build such molecular recognition systems including construction of the molecular recognition moieties on one-dimensional nanoscale materials, such as molecular imprinted nanowires13,14 or nanotube membranes.15 The challenge is to find structures with high stability, high affinity, and low diffusion time of guest molecules. Layer-by-Layer (LbL) self-assembly16 of polyelectrolytes of alternating charge is a powerful technique to construct layered nanostructures with predesigned compositions and tailored functionalities.17-20 As a soft and strongly hydrated material, such polyelectrolyte multilayers (PEM) provide pathways for molecular diffusion and incorporation; for example, PEM are permeable even for polymers21-23 and can take up small molecules by adsorption.24 A certain porosity and heterogeneity of the layer arrangement has been demonstrated both on the nanometer scale as well as on the micrometer scale, respectively.25,26 Taking advantage of the LbL technique, different types of molecules and functional moieties could be incorporated into a nanostructured (13) Yang, H. H.; Zhang, S. Q.; Tan, F.; Zhuang, Z. X.; Wang, X. R. J. Am. Chem. Soc. 2005, 127, 1378. (14) Xie, C. G.; Zhang, Z. P.; Wang, D. P.; Guan, G. J.; Gao, D. M.; Liu, J. H. Anal. Chem. 2006, 78, 8339. (15) Wang, H. J.; Zhou, W. H.; Yin, X. F.; Zhuang, Z. X.; Yang, H. H.; Wang, X. R. J. Am. Chem. Soc. 2006, 128, 15954. (16) Decher, G.; Hong, J. D.; Schmitt, J. Thin Solid Films 1992, 210, 831. (17) Zhang, X.; Shen, J. C. Adv. Mater. 1999, 11, 1139. (18) Tang, Z. Y.; Wang, Y.; Podsiadlo, P.; Kotov, N. A. Adv. Mater. 2006, 18, 3203. (19) Rusu, M.; Kuckling, D.; M€ohwald, H.; Sch€onhoff, M. J. Colloid Interface Sci. 2006, 298, 124. (20) Zhang, X.; Chen, H.; Zhang, H. Y. Chem. Commun. 2007, 1395. (21) Sukhorukov, G. B.; Antipov, A. A.; Voigt, A.; Donath, E.; M€ohwald, H. Macromol. Rapid Commun. 2001, 22, 44. (22) Georgieva, R.; Moya, S.; Hin, M.; Mitlohner, R.; Donath, E.; Kiesewetter, H.; M€ohwald, H.; B€aumler, H. Biomacromolecules 2002, 3, 517. (23) Choudhury, R. P.; Galvosas, P.; Sch€onhoff, M. J. Phys. Chem. B 2008, 112, 13245. (24) Choudhury, R. P.; Sch€onhoff, M. J. Chem. Phys. 2007, 127, 234702. (25) Vaca Chavez, F.; Sch€onhoff, M. J. Chem. Phys. 2007, 126. (26) Wende, C.; Sch€onhoff, M. Langmuir [Online early access] DOI:10.1021/ la904763j, 2010.

Published on Web 03/24/2010

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Scheme 1. Chemical Structure of DAR, Poly(acrylic acid), and Por

thin film, realizing their controlled loading and release. There are some first efforts aiming to employ the LbL technique to solve the problem that remains in the traditional molecular imprinted polymers. For example, Kunitake et al. used the surface sol-gel method to introduce imprinted sites into the inorganic-organic hybrid films.27 The hybrid film is rather rigid, and the selectivity is low. Kanekiyo et al. imprinted AMP into LbL films; however, without cross-linking the stability is low.28 Stable LbL films for molecular imprinting were demonstrated by covalently cross-linking the LbL film after film formation with an included template, and the reversibility of uptake and release was demonstrated on a simple system using UV-vis spectroscopy:29 It was shown that this approach yields imprinted sites in robust multilayer structures, though for the simple Coulomb interactions employed, the selectivity needs further improvement, and the mechanisms and conditions under which such imprinting is feasible require further study. In the present paper, we monitor the construction of such crosslinked LbL films including template molecules and the binding and release of the guest molecules in the imprinted sites employing a quartz crystal microbalance with dissipation measurement (QCM-D). It is found that pH-dependent removal and rebinding of the template leads to a simultaneous swelling of the film. A separation of mass changes induced by swelling from the template uptake/removal is carried out by careful interpretation of the obtained QCM-D data, i.e., by quantifying surface coverage values of the molecular constituents. Finally, the mechanism of pH-driven template uptake and release is demonstrated and interpreted.

Materials and Methods Materials. The following chemicals were used as supplied: Poly(acrylic acid) (PAA) of different molecular weight was obtained as aqueous solution from Sigma Aldrich (Mw = 2000 g/mol (63 wt %), 5000 g/mol (50 wt %), and 100 000 g/mol (35 wt %)). Poly(ethylene imine) (PEI) was obtained from Sigma Aldrich (Mw = 50 000-60 000 g/mol, 50 wt % in water). 5,10,15, 20-Tetrakis-(4-(trimethylammonio)-phenyl)-21H, 23H-porphine tetratosylate (Por) was obtained from ACROS Chemical Company (solid, purity 94%). The photosensitive polycation diazo resin DAR (solid, Mn = 2640) was kindly provided by Prof. Yuping Dong (Beijing Institute of Technology). The relevant components are shown in Scheme 1. Ultrapure water (Milli-Q, 18 MΩ cm) was used in all solutions and for all washing steps. HCl and NaOH were used as 0.1 M solutions from Riedel-de-Haen. UV-vis spectra on coated quartz substrates were performed by a SPECORD PC 50 spectrometer (Analytik Jena) using 4.8 cm  (27) Lee, S. W.; Ichinose, I.; Kunitake, T. Langmuir 1998, 14, 2857. (28) Kanekiyo, Y.; Inoue, K.; Ono, Y.; Sano, M.; Shinkai, S.; Reinhoudt, D. N. J. Chem. Soc., Perkin Trans. 2 1999, 2719. (29) Shi, F.; Liu, Z.; Wu, G. L.; Zhang, M.; Chen, H.; Wang, Z. Q.; Zhang, X.; Willner, I. Adv. Funct. Mater. 2007, 17, 1821.

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1.4 cm  1 mm quartz substrates. Quartz substrates or gold-coated quartz crystals were cleaned in H2O/H2O2/NH3 solution (5:1:1) heating to ca. 70 °C for 15 min. The substrate/crystal was then washed in ultrapure water and dried in a stream of nitrogen. QCM-D measurements were performed using a Q-Sense E4 (Q-sense, Gothenburg) with four flow modules (QFM 401) and gold-coated quartz sensors (QSX 301, Q-sense). The UVirradiation lamp (NU-8 KL, 40 W, Konrad Bender Laborger€ate) provided a UV emission at 254 and 366 nm. Solutions. The PAA and DAR solutions were prepared with 1 mg/mL concentration, different molecular weight in case of PAA are denoted as PAAMw. The PEI solution had a concentration of 10 mM monomer concentration. The precursor complex was prepared by adding 5 mL of Por solution (0.5 mg/mL) to 25 mL of PAA solution under ultrasonic stirring. The resulting pH value of the polymer solutions was pH ∼3.8 for PAA/Por solution and pH ∼2.7 for DAR solution, and was not further adjusted. UV/vis Spectroscopy. The multilayers were prepared by dipping the cleaned substrate into solutions of polyions of alternating charge, starting always with PEI, until yielding PEI(PAAPor/DAR)5 multilayers. The adsorption time is 20 min with repeated washing in ultrapure water between layers (4  1 min). After 11 layers, the spectrum was recorded and UV irradiation was done for 8 min from a distance of ∼10 cm, followed by recording another spectrum. Release and rebinding of the template was carried out by dipping the substrate into HCl solution (pH = 1) and Por solution (0.5 mg/mL, pH = 7-10), respectively, with subsequent washing. From the absorbance band of DAR, the surface coverage ΓDAR was determined: The band was fitted with a model curve and the integral of the signal was compared to external calibration data of DAR in solution. The surface coverage of Por, ΓPor, was determined in the same way after subtraction of the DAR absorption spectrum. Quartz Crystal Microbalance. A dissipative quartz crystal microbalance (QCM-D) with flow cells for in situ experiments was employed (Q-sense, E4). Polyelectrolyte multilayer (PEM) deposition on the surface of gold-coated quartz was achieved by flowing polyelectrolyte solutions (1 mg/mL) in an alternating manner (PEI(PAA-Por/DAR)5) through the QCM-D flow cell (0.1 mL/min) using water as washing agent in between polymer solutions. The first layer was always poly(ethyleneimine) (PEI, 10 mM). The deposition time for each layer was 20 min; the washing time was 10 min. The photo-cross-linking was accomplished by removing the coated quartz sensor from the flow module and irradiating it for 8 min using a UV-lamp (distance to sample ∼10 cm). Thereby, the covalently cross-linked surface molecular imprinted layer-by-layer films (SMILbL) were obtained. After reinserting the sensor into the device, release and rebinding of the template was carried out by flowing HCl solution (pH = 1) and Por solution (0.5 mg/mL, pH = 7-9) through the module, again with intermediate washing steps. The QCM-D device records the change of the resonance frequency (Δf) of the quartz with time. The relation between Δf and the change of mass at the surface (Δm) is given by the Sauerbrey equation30 Δf ¼ - 2fG2

Δm FG νG

ð1Þ

The constants fG, FG, and νG depend on the properties of the used quartz. The QCM-D also records the change of dissipation (ΔD) during the measurement. ΔD is defined as dissipation (loss) of energy during one oscillation period of the quartz due to the viscoelastic properties of the adsorbed material. It is D ¼

Ediss 2πEsto

ð2Þ

(30) Sauerbrey, G. Z. Phys. 1959, 155, 206.

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where Ediss is the energy loss, and Esto represents the energy stored by the oscillator during one oscillation period. The obtained values for ΔD are on the order of magnitude of 10-6. Multilayers with ΔD < 2  10-6 are rigid and follow the Sauerbrey equation. For systems with larger dissipation, correction terms for the Sauerbrey equation are needed to take the viscoelastic properties into account.31 The raw data typically show a decrease in Δf while polyelectrolyte solution is flowing through the cell, which corresponds to an increasing amount of adsorbed mass at the surface. When Δf reaches a constant value (plateau), the saturation coverage of the surface is reached. During the washing steps, loosely bound polymer chains typically desorb and Δf slightly increases. Successful multilayer buildup is therefore detected by an increasing Δf and saturation coverage for each layer, which leads to a plot with a “step” for each layer. The frequency change after n layers (including washing) is denoted as Δfn; the frequency change of one single layer is therefore presented as Δfn - Δfn-1.

Results and Discussion 1. Molecular Weight Dependence of Imprinted Layer Formation. To obtain complexation of the template with PAA, the charge of the polymer chain is an important parameter. Here, we study the role of the number of charges per chain by using PAA of different molecular weight. UV-vis spectroscopy can prove the presence of DAR and Por by their absorption wavelength and allows the calculation of the surface coverage after each adsorption step. Figure 1a shows the absorption spectrum of multilayers prepared from DAR/PAA100k-Por, demonstrating the incorporation of both DAR and Por by the presence of the two maxima at 380 and 420 nm, respectively. Figure 1b,c gives the surface coverage Γ of either component in dependence of the number of layers deposited, proving a regular layer of buildup for all molecular weights of PAA employed, i.e., 100 kDa, 5 kDa, and 2 kDa. Note that the increase of the DAR absorbance/surface coverage occurs with the deposition of the odd layer numbers (DAR layers), while the Por increases with the adsorption of each even layer number (PAA layers), as expected. In the case of PAA2k, no Por adsorption was detected, and no difference of the DAR surface coverage was induced by the presence of Por in the PAA solution. The surface coverage of the template shows a significant decrease after adsorption of each odd layer. This indicates a competitive adsorption of the cationic species Por and DAR to the outermost PAA layer, where the template is partially replaced by the polycation. This applies for both different molecular weights, however, the amount of bound template is nevertheless satisfying after deposition of 11 layers. In contrast to molecular surface coverage, the QCM-D measurements determine the total adsorbed mass on the surface during layer formation. Figure 2 shows the development of the mass coverage during multilayer buildup for imprinted and not imprinted multilayers from PAA100k, respectively. A successful stepwise buildup of the multilayers is demonstrated by each adsorption step leading to an increase of mass coverage. The multilayers built from Por-containing polymer solution (Figure 2a) show generally higher values of (Δfn - Δfn-1) for PAA100k layers, as compared to the not imprinted film (Figure 2b). This is a strong indication for the inclusion of the template, whereas the (Δfn - Δfn-1) values of the DAR layers are similar in both systems. The multilayer buildup using PAA2k-Por (data not shown) causes frequency changes that match those of not

imprinted layers of PAA100k. This indicates the absence of the template due to insufficient binding in agreement with the results from the UV-vis spectra. The measurements with PAA5k (data not shown) show the inclusion and absence of the template with basically the same properties as films made from PAA100k. This is in agreement with the UV-vis spectra for PAA5k and PAA100k films, which showed a very similar layer buildup. It was previously shown that basic conditions (pH = 12) lead to a sufficiently charged PAA2k chain which can bind Por and allow SMILbL formation.32 However, we found that, due to the sensitivity of DAR, which is decomposing during the adsorption of the PAA-Por layers from basic solution, high pH values do not provide suitable preparation conditions. Here, we have demonstrated that SMILbL formation at lower pH values becomes

(31) Vogt, B. D.; Lin, E. K.; Wu, W. L.; White, C. C. J. Phys. Chem. B 2004, 108, 12685.

(32) F€orster, T. In Biological Physics, Mielczarek, E. V., Greenbaum, E., Knox, R. S., Eds.; American Institute of Physics: New York, 1993; pp 183-221; Translation of F€orster, T., 1948; 1993.

10124 DOI: 10.1021/la1002447

Figure 1. (a) Absorption spectra of n layers containing DAR and PAA100k-Por. (b) Calculated surface coverage ΓDAR dependent on the number of layers for PEM using PAA100k imprinted (filled squares), not imprinted (open squares); PAA5k imprinted (filled circles), not imprinted (open circles); and PAA2k not imprinted (open triangles). (c) Calculated surface coverage ΓPor in imprinted films using PAA100k (squares) and PAA5k (circles).

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Figure 2. Frequency and dissipation change from QCM-D measurements of the multilayer formation of (a) imprinted layers, PEI(PAA100k -Por/DAR)5, and (b) not imprinted layers, PEI(PAA100k/DAR)5. The inserts show the net frequency change due to the adsorption of a single layer. Table 1. Charges per PAA Chain for Different Chain Length and Average Surface Coverage per DAR Monolayer Extracted from UV/vis and QCM Data, Respectivelya imprinted film Mw/g monomers charges ΓDAR/ Γ*DAR/ mol-1 per chain per chain mg m-2 mg m-2

not imprinted film ΓDAR/ mg m-2

Γ*DAR/ mg m-2

2000 28 2 6.6 2.4 (4.4) 5000 69 5 7.6 2.8 (8.0) 6.5 2.2 (4.6) 100 000 1389 97 7.5 2.0 (10.0) 6.3 2.6 (5.4) a Values with ‘*’ result from QCM-D and include the water contained in the film. Values in brackets give the surface coverage per bilayer.

feasible, if longer chains are employed, such that they provide sufficient charges. A surface coverage Γ*DAR for each single DAR layer, including bound water, can be determined from the QCM-D measurements by using the Sauerbrey equation, since the dissipation is low in all cases. Using the known properties of quartz, the Sauerbrey equation is Δm = -CΔf, with C = 17.7 ng Hz-1 cm-2. Surface coverage values are shown in Table 1, which is summarizing data for the coverage per DAR layer from UV/vis data, ΓDAR, and the total mass coverage per DAR layer (including water), Γ*DAR, determined from QCM-D data. The successful binding of the template to PAA5k and PAA100k in contrast to PAA2k can be explained by the degree of charge of PAA: Choi and Rubner determined the ratio of dissociated monomers per PAA chain in solution at different pH values: At pH = 4, about 7% of the monomers are dissociated and thereby charged.33 The resulting number of charges per chain for each (33) Choi, J.; Rubner, M. F. Macromolecules 2005, 38, 116.

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molecular weight employed in this work is given in Table 1. About five charges per chain are apparently needed for sufficient binding of Por by PAA in solution. Since Por has four positive charges, this means that at least the number of template charges needs to be present on one single chain in order to cause complexation and successful deposition of template. Additional potential mechanisms such as a complexation of a Por molecule with several chains, or an increase of the degree of dissociation of the chains in the presence of Por seems to be not relevant. The Γ values are of the same order of magnitude as determined for PEM from typical strong polyelectrolytes.34 Comparing the values for imprinted and not imprinted films, the surface coverage of a DAR/PAA bilayer is generally higher for imprinted films. The Γ*DAR values do not vary significantly in the different multilayer systems; they are nearly constant between 2 and 2.8 mg/m2. This leads to the conclusion that the imprint takes place in the PAA layers due to their opposite charge, while the DAR is mainly important for the LbL assembly and the later cross-linking, but does not support, nor is influenced by, the template binding. An interesting result is the difference between ΓDAR and Γ*DAR. Despite the fact that Γ*DAR includes the water in the layer, its values are lower than those of ΓDAR, which do not contain the water contribution. The explanation for this might be different preparation conditions for the UV/vis experiments as opposed to QCM-D: First, the layer assembly prepared under flow might lead to lower adsorption than the standing solutions used in the dipping for UV/vis spectroscopy. Though such effects have been observed,35 however, the layer thickness shows only minor dependencies on flow velocity and cannot explain the factor of 3 between ΓDAR and Γ*DAR. Second, the drying process involved in layer preparation for UV-vis measurements might lead to different adsorbed amounts as compared to never-dried films, which were employed in QCM-D experiments. Again, it is unlikely that the difference would amount to a factor of 3. In conclusion, it is the water contained in the quantity Γ*DAR which is responsible for its low value: Upon deposition of the DAR layer, water is released from the film, giving a negative contribution to Γ*DAR and decreasing it far below the value of ΓDAR. It is interesting to note that the bilayer mass coverage, given in brackets, is always roughly on the order of ΓDAR. This suggests that upon DAR adsorption water is released from the film, while PAA or PAA/Por adsorption causes water uptake. It can be expected that, after each bilayer deposition cycle, the hydration state of the total film is reproduced again. It has to be noted, however, that, due to possible differences in multilayer formation from different stirring or flow geometries, the UV-vis and QCM-D data are not in detail quantitatively comparable. For example, the fact that for the not imprinted film the total bilayer coverage is even slightly lower than the DAR coverage, i.e., Γ*DAR, bilayer < ∼ΓDAR can only be explained by differences due to deposition with and without flow, respectively. 2. Photo-Cross-Linking. To observe the photo-cross-linking and the release/rebinding, SMILbL made with PAA100k were chosen because they showed a regular layer buildup with good Por incorporation. The irradiation with ultraviolet radiation leads to cross-linking of the film by release of nitrogen from the diazonium moiety (Scheme 2). Therefore, the UV-vis spectra show that the absorbance at 380 nm disappears after irradiation, while the absorbance of the Por remains (data not shown; see ref 29). With the DAR absorbance band no longer present in the spectrum, the calculation of the surface coverage from the (34) Xie, A. F.; Granick, S. Macromolecules 2002, 35, 1805. (35) M€uller, M. Personal communication, 2009.

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Scheme 2. Formation of Cross-Linking Ester Bonds upon UV-Irradiation

Figure 3. Change of frequency and dissipation during film treatment with 0.1 M HCl: black lines, imprinted film; gray lines, not imprinted film. Table 2. Por Surface Coverage ΓPor Occurring upon Rebinding of Por at Different pH Values Extracted from UV/vis and QCM Data, Respectivelya imprinted film pH

template absorption is straightforward, resulting in 4 mg/m2 for the 11 monolayer system. This value is close to ΓPor from the analysis of spectra before cross-linking, employing the subtraction of the DAR band, where it is ΓPor = 5.5 mg/m2 (see Figure 1c). It appears that there is a slight loss of Por during cross-linking. To cross-link the layers in QCM-D measurements, the flow cell has to be opened to remove the quartz sensor. For this reason, QCM data before and after cross-linking cannot be compared. 3. Template Release. The removal of the template can be realized by treatment of the multilayers with HCl solution (0.1 M). The feasibility was demonstrated previously by UV-vis spectroscopy.29 In acidic conditions, the carboxylate moieties are protonated and disable the electrostatic complexation of the positively charged template to negative polymer charges. Similarly, in the present films a full release of the template is demonstrated in UV/vis spectra by a complete disappearance of the Por resonance (data not shown). Here, we study changes of the surface coverage and viscoelasticity upon acid treatment by QCM-D in order to investigate the mechanism of the template release in acid conditions. The released template is clearly visible in the solution leaving the cell, detected by a characteristic green color. After ∼8 min of flowing with HCl, the solution is clear again, marking the complete release of the template from the multilayers. Thus, under these flow conditions, the release is much faster as compared to samples for UV/vis experiments kept in static conditions.29 Figure 3 shows the changes of Δf and ΔD occurring upon treatment with HCl. The QCM-D registers an increase of mass (decreasing Δf) during the release of the template. This is surprising, since the loss of template would cause a decrease of the mass coverage. The mass increase can be explained by water uptake of the multilayers and pH-induced swelling of the film: Proton uptake into the multilayer will change the charge equilibrium. Though proton uptake can be partially compensated by Por4þ release, additional protonation can produce a net positively charged, self-repulsive layer, causing hydration water uptake to enable screening of the charges. In detail, under acid conditions, the amine moieties of the 10126 DOI: 10.1021/la1002447

ΓPor/mg m

-2

not imprinted film

Γ*Por/mg m

-2

water 2.7 3.3 9 11 10 12 17 a Values with ‘*’ result from QCM-D.

ΓPor/mg m-2

Γ*Por/mg m-2

1.5 3.0

3.2 8.5 11

DAR can become protonated, and in addition free carboxylate groups in the film, which are neither complexed with Por charges, nor have reacted with DAR, might be neutralized by protonation. Both protonation reactions can contribute to overcharging with positive charges and thus cause film swelling due to the selfrepulsion of the polymer network. In this context, it is important to note that, though dissociation equilibria in multilayers are modified as compared to the same chains in solution, for example pKa values shift,36 weak polyions in multilayers can still be protonated and deprotonated. A pH-driven uptake and release in polyelectrolyte multilayers made of weak polyelectrolytes has already been shown previously for single and double charged dyes.37 In a number of further publications, pH-dependent swelling is documented.38,39 In comparison to the not imprinted system, the imprinted film shows much larger water uptake and larger viscoelasticity. This can be attributed to a higher porosity and lower cross-linking density of the polymer network caused by the inclusion of the template. 4. Rebinding of the Template. The rebinding of the template is done using Por solution (0.5 mg/mL) adjusted to different pH values. Table 2 shows the results of rebinding of Por performed at different pH conditions. It can be seen that, even in the nonimprinted film, Por uptake takes place. This can be considered as unspecific binding of Por. On the other hand, the film with imprinted binding sites leads to a higher Por uptake. It is interesting that this difference is particularly pronounced at the higher pH value of 10, since it suggests that the unspecific binding has a less pronounced dependence on pH, while specific binding is strongly pH driven. This is in agreement with the binding concept pursued here, i.e., the (36) Rmaile, H. H.; Schlenoff, J. B. Langmuir 2002, 18, 8263. (37) Burke, S. E.; Barrett, C. J. Macromolecules 2004, 37, 5375. (38) Kim, B. S.; Vinogradova, O. I. J. Phys. Chem. B 2004, 108, 8161. (39) Mauser, T.; Dejugnat, C.; Sukhorukov, G. B. Macromol. Rapid Commun. 2004, 25, 1781.

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Figure 4. Frequency change and dissipation change upon consecutive film treatment with I, ultrapure water; II, water (pH 9); and III, Por solution (pH 9).

formation of pH-dependent imprinted binding sites. It can be concluded that the deprotonation of the PAA acid functions in the imprinted binding sites is the driving force of the specific part of Por rebinding. Even when taking into account that the imprinted film has a larger surface coverage and thickness than the nonimprinted one (see coverage data of Table 1), it is still more than 50% of the bound Por that is specifically bound in the binding sites. The results of Δf show an increase of the mass coverage with Por binding. This might appear surprising, since the opposite process, the Por release, also leads to film swelling. The reason for this swelling can be seen in the fact that an electrostatically well balanced film, equilibrated in pure water, is now subject to a solution of nonzero ionic strength. It has been shown previously that salt solutions lead to swelling of PEM, though saturation of the swelling already occurs at low concentration, and only a low fraction of salt actually incorporates into the film.40 The same effect applies here, which is consistent with the small amount of swelling and very similar behavior of imprinted and not imprinted film. Treatment with neutral template solution leads again to unspecific binding for both kinds of films. The imprinted film is increasing strongly in mass upon rebinding of template from basic solution. This is of course due to rebinding of the template itself, while further electrostatic loading of the film due to deprotonation, as well as hydration water, probably also contribute. For the higher pH values, Por uptake is much more strongly pronounced, and in addition more dependent on the existence of imprinted sites. An interesting question is how far treatment with a basic solution alone would already cause film swelling. Therefore, a consecutive experiment was performed (see Figure 4). Following template release, after washing of the film with water (region I) the film is first treated with basic NaOH solution of pH 9 (region II), and only in a later step (region III), the film is in contact with a Por-containing solution of pH 9. Apparently, pH treatment alone cannot change the Δf value and lead to film swelling. Only the template solution at pH 9 leads to an increase of mass coverage, which is caused only by the rebinding of the template. Since it was shown before that the mass decrease due to loss of Por is small compared to mass changes caused by hydration water, the swelling occurring at the beginning of region III can be as attributed to swelling predominantly by water, while the mass of Por is negligible. The film is thus swelling with water; however, (40) Sukhorukov, G. B.; Schmitt, J.; Decher, G. Ber. Bunsen-Ges. Phys. Chem. 1996, 100, 948.

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Article

Figure 5. Variation of the resonance frequency in a cyclic treatment starting after cross-linking: black, imprinted film; gray, not imprinted film. Down arrow, injecting solution of pH = 1 for release; w, washing with water at neutral pH; up arrow, injecting Por solution (pH 9; third cycle, pH 10).

this swelling has to be induced by the presence of Por in the solution. After increase of the pH to 9, the system can be considered frustrated in its dissociation equilibrium: Deprotonation would be induced at high pH; however, there is no charge regulation possible due to the lack of mobile charges. Only upon Por addition can deprotonation occur and the charge equilibrium is restored by Por incorporation into the film. The strong swelling in the presence of Por again points at a self-repulsive filml; probably, the 4-fold positive charge of Por acts in a cooperative way to cause up-charging. 5. Cyclic Release and Rebinding. The frequency change during repeated binding, washing, and release cycles was monitored in a long-term measurement (Figure 5). At the beginning of the experiment, the template is released with acidic solution and the swelling is easy to monitor. Following this, when treating the film with water as washing solution, deswelling of the film occurs and the frequency increases to a Δf value of -120 Hz for the imprinted film. This is explained by the higher pH of the water, which reduces the amount of charged groups in the film, which were the initial reason for swelling. Therefore, washing results in a compact conformation of the polymer network under the influence of the higher pH of water. This conformation still contains more water than the freshly cross-linked film, which was calibrated as Δf = 0, which indicates that the initial swelling is not reversible. This behavior is also found for the not imprinted film. The template uptake is clearly visible by the decrease of the frequency upon treatment with template solution. The repetition of the procedure two more times shows that the swelling/deswelling steps after the initial swelling are reversible; the film is oscillating between two equilibrium states. An interesting feature is the small peak, occurring reversibly upon the second and any further release steps. An enlargement of this peak is shown in Figure 6. It occurs for both imprinted and not imprinted film, but with larger amplitude for the imprinted film. The film mass is reduced for about one minute, until the reduction is superimposed by the film swelling, increasing the mass coverage. Since the time scale matches well with the time of several minutes for the direct observation of colored solution (see further above), it is likely that this peak marks the release of Por, which occurs-at least partly-somewhat faster than film swelling, the latter taking several hundred minutes (see Figure 5). 6. Mechanism of Por Uptake and Release. The above findings show that a reversible uptake and release of Por can be accomplished in the multilayer imprinting system. The mechanism is based on the pH-driven protonation and deprotonation of the weak polyelectrolyte component PAA. In particular, the DOI: 10.1021/la1002447

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charge, since Por is adsorbing into the film. On the one hand, the Por charges might just replace the protons of protonated PAA groups in the binding sites; in this case, the charge equilibrium would not be affected. However, due to the large amount of swelling, an overcharging due to Por incorporation has to be postulated. This overcharging might arise through Por molecules binding with only two or three of their positive charges to opposite charges in the film, thus introducing excess positive charge.

Conclusions

Figure 6. Change in frequency at the beginning of HCl treatment of the imprinted (black) and not imprinted (gray) film.

Figure 7. Mechanism of reversible binding and release.

strong swelling observed in QCM-D experiments is a signature of water uptake into the film and can be taken as an indication for charging and self-repulsion of the film. Thus, as shown in the sketch in Figure 7, Por release in acid conditions is accompanied by film swelling, since the Por release is mainly driven by protonation and overcharging with positive charges. Washing with water or basic solution (3rd image in Figure 7) causes deswelling, since at neutral pH, the protonation is decreased and the charge equilibrium in the film is restored. However, when Por is offered again in the solution, swelling occurs even without a pH change in the solution (4th image in Figure 7). This can be attributed to an overcharging with negative

10128 DOI: 10.1021/la1002447

The multilayer buildup and the release/rebinding of the template can be performed and observed successfully by means of the quartz crystal microbalance, with the process being confirmed by UV-vis experiments. The usage of higher molecular mass poly(acrylic acid) allowed template binding and multilayer buildup at low pH value of 4. During the preparation of the imprinted layers the QCM-D yielded information about the surface coverage of the layers as well as the total surface coverage of the film. By observing the template kinetics on a second-to-minute time scale, it was found that pH-induced water uptake and swelling superimposed the release of the template. Template release is clearly driven by protonation and overcharging of the film. The rebinding of the template works in basic conditions by charging the binding sites due to deprotonation. The presence of imprinted binding sites is indirectly proven by comparing the change in mass and viscoelasticity with the not imprinted system. The major part of template uptake at high pH is due to the presence of imprinted sites, while only a minor fraction of template binds unspecifically into the film. Acknowledgment. This project is funded by the Deutsche Forschungsgemeinschaft (DFG) within the Chinese-German collaborative research center TR 61 ‘Multilevel Molecular Assemblies: Structure, Dynamics and Function’, project number B11. We thank Prof. Yuping Dong (Beijing Institute of Technology) for kindly providing the DAR azo resin polymer.

Langmuir 2010, 26(12), 10122–10128