Cucurbit[8]uril as Nanocontainer in a Polyelectrolyte Multilayer Film

Several fundamental steps are required for the cycle of guest molecule uptake and release to occur in a fast and reversible manner: first, recognition...
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Cucurbit[8]uril as Nanocontainer in a Polyelectrolyte Multilayer Film: A Quantitative and Kinetic Study of Guest Uptake Henning Nicolas,† Bin Yuan,‡ Jiawei Zhang,‡ Xi Zhang,‡ and Monika Schönhoff*,† †

Institute of Physical Chemistry, University of Muenster, Corrensstrasse 28/30, 48149 Münster, Germany Key Laboratory of Organic Optoelectronics & Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China

‡

S Supporting Information *

ABSTRACT: The host−guest chemistry of cucurbit[8]uril (CB[8]) and the layer-by-layer self-assembly technique are combined to obtain a molecular imprinted polyelectrolyte multilayer film for the recognition and binding of a guest molecule. Cucurbit[8]uril as a ready-made binding site is first associated with a polyelectrolyte and then assembled into a polyelectrolyte multilayer film via layer-by-layer deposition. A cationic guest is subsequently included into the nanocontainer due to specific host−guest interactions. The quantitative analysis of both CB[8] and the included guest molecule in dependence of the surface charge of the multilayer film identifies a high nanocontainer density as well as good to excellent binding efficiencies, therefore yielding a promising imprinted nanomaterial with potential applications in filtration or sensor technology. The investigation of the guest molecule uptake kinetics reveals two processes on different time scales, respectively, which are again related to the charge of the multilayer film surface. The combination of the results obtained from both ultraviolet spectroscopy and dissipative quartz crystal microbalance enables us to describe a full picture of several simultaneous processes initiated by the guest molecule.

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system is polyelectrolyte multilayer films (PEM), which are easily fabricated via the alternating deposition of oppositely charged building blocks by following the protocol of the layerby-layer self-assembly technique (LbL).10−12 In 2007, Shi and co-workers successfully combined molecular imprinting in polymeric matrices with unconventional LbL self-assembly and thus established the novel concept of surface molecular imprinted polyelectrolyte multilayer films (“SMILbL”),13 leading to a thin material of only a few nanometers in thickness which is capable to perform a rapid uptake and release of a guest molecule. In general, the preparation of such surface molecular imprinted LbL multilayer films is based on a three-step procedure. First, the template molecule and a macromolecular compound are preassembled due to intermolecular interactions, therefore obtaining a stable complex in solution which is subsequently used for the fabrication of a multilayer film. So far, several types of interactions, i.e., hydrogen bonds,14 electrostatics,13,15 or host−guest interactions,16 have successfully been used for the precomplex formation in different SMILbL approaches. As second step, alternating LbL deposition of the preassembled macromolecular complex and a corresponding building block incorporates the template molecule into a multilayer film. In several SMILbL approaches, a subsequent cross-linking is

INTRODUCTION The selective binding and release of a guest molecule performed by a recognizing entity are decisive processes in a variety of natural host−guest systems. Proteins, receptors, membranes, and antibodies are only a few of numerous examples wellknown for the formation of biologically active host−guest complexes. Several fundamental steps are required for the cycle of guest molecule uptake and release to occur in a fast and reversible manner: first, recognition and binding of the guest molecule by the host due to attractive interactions and secondly the release from the hosting cavity as a result of a particular trigger, e.g., a change of the chemical environment. In between, the host might be executing a certain task on the guest molecule such as a structural modification, for instance in terms of catalysis, separation, or simple transportation.1−3 At last, the binding site must remain undamaged to perform the next uptake and release cycle.4,5 By means of mimicking these unique key processes of nature, the concept of molecular imprinting in cross-linked polymer materials (MIP) was described for the first time by Wulff, presenting a protocol to functionalize polymeric matrices with artificial binding sites.6−9 However, as a consequence of their macroscopic character, it turned out that MIP are suffering from elongated diffusion pathways as well as a high number of buried binding sites and therefore a low binding efficiency. Challenged by these facts, researchers transferred the molecular imprinting approach to nonbulky, planar nanostructures. A suitable basis as a well-established and controllable nanolayer © 2015 American Chemical Society

Received: July 30, 2015 Revised: September 15, 2015 Published: September 15, 2015 10734

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Figure 1. Representation of (1) the uptake of the linear guest molecule into the imprinted nancontainers followed by (2) the release of the guest due to a chemical treatment. After this, the nanocontainers are recovered (3) and can be used for another reversible uptake and release cycle.

host−guest chemistry. Each representative of CB[n], often declared as “pumpkin-shaped molecules” with respect to their particular macrocyclic structure, consists of n glycoluril monomers which generate an inner cavity of different sizes.25−30 As the key player, the cavity of CB[8] is capable to include either one or two guest molecules, meaning that both binary and ternary complexes can be formed. It is required that the molecular geometry of the hosted compounds allows the vicinal alignment. The rims of CB[8] are terminated by two portals formed by the carbonyl groups of glycoluril and therefore act as electrostatic dipoles, making the incorporation of cationic organic ammonium compounds favorable due to ion-dipole interactions.26,31−35 To date, a long list of guest molecules has been found to form stable host−guest complexes with CB[8].36 In this work, we expand the research on the novel approach using CB[8] in SMILbL by studying quantitative and kinetic characteristics of the guest molecule uptake and release (Figure 1). We address the amount of imprinted nanocontainer within the PEM as well as the binding efficiency of CB[8] in this environment. We employ two isomeric guests with either a linear shape (“AnPy”) or a branched shape (“9-AnPy”) toward the nanocontainer in the polyelectrolyte multilayer film. While the linear guest fits into the cavity of CB[8], the branched isomer is excluded due to sterical hindrance. The comparison of guest molecules that are binding or nonbinding into the container, respectively, allows to discern selective and unselective binding processes in the film material. Apart from quantitative uptake data, we also study the kinetics of the uptake process, which sheds more light on the underlying mechanisms of CB[8]−guest complex formation in the sterically restrictive environment of the polymer material.

required as the key step to provide a stable matrix with binding sites. Finally, the template can be removed from the polymer matrix by disabling the attractive interactions, and therefore the imprinted PEM becomes capable of performing reversible uptake and release cycles due to specific interactions toward the template. Since the pioneering work of Shi et al., SMILbL was extended with respect to the interactions used for the uptake and release mechanism, improving binding efficiencies and selectivity.17 In addition, it was further brought to colloidal particles to enhance the surface area,18,19 SMILbL systems for protein recognition were developed,19 cross-linking and preparation strategies were reported,20,21 and finally SMILbL demonstrated potential for further application, e.g., in sensor devices22 or in selective filtration achieved by attaching an imprinted film to porous membranes.23 A recent review of current developments in molecular imprinting is given by Whitcombe et al.24 In a very recent approach, Zhang et al. incorporated cucurbit[8]uril (CB[8]) as a nanocontainer in a PEM via its precomplexation with a tailor-made polyelectrolyte. After that, they demonstrated the successful recognition of a guest molecule followed by the inclusion into the nanocontainer due to specific host−guest interactions. Furthermore, the subsequent release of the guest molecule from the nanocontainer was executed and enabled several reversible uptake and release cycles.16 It is important to note that the assembly of CB[8] into multilayer films only loosely relates to a SMILbL approach in terms of the classic definition of molecular imprinting, since the formation of an effective “imprint” of binding sites is no longer required. Since CB[8] was locked into the multilayer films in our previous work by a photochemical cross-linking, the novel procedure might rather be described as an incorporation of host molecules in multilayer films. The class of cucurbit[n]urils (CB[n]) attained remarkable attention within the past decade due to its great potential in

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MATERIALS AND METHODS

Materials. Poly(ethyleneimine) (PEI, Mw = 50 000−60 000 g mol−1, 50% wt in water), poly(acrylic acid) (PAA, Mw = 100 000 g mol−1, 20% wt in water), cucurbit[8]uril hydrate (CB[8], Mw = 1329.12 g mol−1), 10735

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material directly from Sauerbrey’s relation (eq 1),38 which holds for sufficiently thin and rigid films.

NaOH (0.1 M), and HCl (0.1 M) were purchased from Sigma-Aldrich, and sodium borohydride was from Acros Organics. A statistical copolymer bearing a viologen side chain (PMV) was synthesized according to previous protocols16 by radically copolymerizing (vinylbenzyl)trimethylammonium chloride and 4-vinylbenzyl chloride, reacted with butylviologen, yielding the structure of PMV displayed in Figure 2. Guest molecules (AnPy, 9-AnPy) were synthesized according to the protocols published before.16

Δf = − Cf Δm

Hz cm 2

(1)

Cf is the sensitivity constant which depends only on the material properties of the quartz sensor. The sensors employed had a resonance frequency of f 0 = 4.95 MHz with Cf = −17.7 ng Hz−1 cm−2. QCM-D is also able to simultaneously monitor the change of dissipation (ΔD), therefore providing information about the viscoelastic properties of the deposited material. D is defined as the ratio of the dissipated energy Ediss to the stored energy Estored in a single oscillation; thus, ΔD yields information about the viscoelastic properties. ΔD is also important to judge about the validity of eq 1, which is given as long as ΔD ≤ 2 × 10−6.39,40 In Situ Film Formation in QCM-D. After purification, the QCM-D sensors were carefully mounted into the flow cells. The whole system was flooded and equilibrated with pH-adjusted washing water for at least 30 min. f 0 and overtones were continuously monitored to ensure a constant baseline. In all experiments, PEI was chosen to form the initial layer; therefore, PEI solution was flown through the QCM-D cells at a flow rate of 100 μL min−1. After surface saturation, detected by Δf reaching a plateau, a flow of pure water was used for at least 10 min to remove excess polyelectrolyte. Subsequently, the next polyelectrolyte layer was deposited by following the protocol as described for PEI. In this manner, PAA and PMV-CB[8] were alternately adsorbed until the multilayer film PEI/(PAA/PMV-CB[8])8 with a positive surface charge was obtained. To achieve a negatively terminated PEM, another PAA layer was deposited to create PEI/(PAA/PMV-CB[8])8/PAA. After several minutes of washing with water, the film was exposed to a solution of either AnPy or 9-AnPy, respectively, for several hours. Please note that the release of the guest molecule could not be investigated by QCM-D technique due to the gas development of the reducing agent. As a consequence, gas bubbles attached to the QCM sensor, causing a drastic change of the resonance frequency and therefore making it impossible to obtain reliable data for the release process. Film Preparation by Dip Coating and UV−Vis Experiments. In order to perform UV−vis spectroscopy for quantification, the multilayer films were assembled on quartz substrates by utilizing a programmable dipping robot (DR-1, Riegler and Kirstein, Berlin). The substrates were cleaned as described above. Since the polyelectrolyte solutions were exposed to air for the entire time of film preparation, all compounds used for film preparation guest uptake and release experiments were solved in phosphate buffer (10 mM) to ensure a stable pH value (7.4). The adsorption time was 30 min for all polyelectrolytes. After each adsorption step, the substrates were immersed into three different buffer solutions for 2 min each to remove excess polyelectrolyte. Following the multilayer construction, the film was dried with nitrogen and absorbance was measured to confirm the multilayer formation. A clean quartz substrate was used as a reference. After that we quantified the surface coverage of CB[8] (ΓCB[8] in Table 1) as follows: The polymer PMV has a significant absorbance at ca. λ = 263 nm, which is attributed to the viologen side chain (APMV‑CB[8] in Table 1). This absorbance is slightly reduced when CB[8] is associated (see Results and Discussion). Therefore, we prepared a calibration curve from PMV-CB[8] in solution (see Figure SI-1), which enabled us to calculate the viologen surface coverage within the multilayer film in this way, hereby assuming that the LbL assembly with PAA does not significantly change the absorbance of the CB[8]-carrying viologen side chain. Since the ratio between viologen unit and CB[8] is 1:1, the surface coverage of the viologen is equal to ΓCB[8]. The multilayer films were then stored in buffer overnight to equilibrate the content of hydration water. The molecular uptake was monitored in dependence of time by immersing the substrates in the solution of AnPy or 9-AnPy, respectively. After rinsing and drying, the absorbance was measured against another identical, but unloaded, multilayer film as a reference to quantify the incorporated amount of both molecules. For this, the absorbances of AnPy and 9-AnPy at λ = 357 nm and λ = 371 nm were used, respectively. Absorbance values

Figure 2. Chemical structures of cucurbit[8]uril, polyelectrolytes, and guest and control molecules. Solutions. For QCM-D experiments, solutions of AnPy and 9-AnPy were prepared in Millipore water with a concentration of 1 mM. PEI and PAA were used in a concentration of 10 mM referring to the monomer concentration of the polymers. PMV (1 mM, referring to the viologen unit) was mixed with CB[8] in a molar ratio of 1:1 and then heated to 70 °C for at least 6 h to obtain the binary polyelectrolyte building block PMV-CB[8] (1 mM). For the guest molecule release, a solution of the reducing agent NaBH4 (0.1 mg/mL) was used. For QCM-D experiments, the pH values of all solutions including wash water were adjusted to pH 5 with NaOH or HCl. For dipcoating experiments, all solutions were prepared in phosphate buffer (10 mM) to ensure a stable pH value over the complete period of multilayer film preparation. Cleaning of Substrates. Prior to an experiment, the sensors used for QCM-D measurements were treated for 20 min in RCAsolution (NH3:H2O2:H2O = 1:1:5). After that, they were rinsed with water and dried in a nitrogen flow. The same protocol holds for the quartz substrates used for the preparation of dip-coated multilayer films. Dissipative Quartz Crystal Microbalance (QCM-D). QCM-D (E4, Q-Sense, Biolin Scientific) is a highly sensitive instrument for the in situ monitoring of small surface coverage changes on the order of magnitude of few nanograms.37 For this, a gold-coated quartz sensor is excited at its resonance frequency f 0 by applying an alternating voltage, thus taking advantage of the inverse piezoelectric effect. When additional mass (Δm) is adsorbed on the negatively charged sensor surface, e.g., by the formation of a polyelectrolyte layer, f 0 changes by a value Δf. From Δf one is able to calculate the mass of the adsorbed 10736

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Table 1. Quantification of the CB[8] Imprinting Density ΓCB[8] in the PEM and Both the Incorporated and Released Amounts of AnPy for Three Uptake and Release Cyclesa surface charge of PEM

APMV‑CB[8]

ΓCB[8] (μmol cm−2)

negative

1204

62

AnPy

positive

1203

62

AnPy

negative positive

1218 1210

62 62

9-AnPy 9-AnPy

molecule

Γguest after uptake(μmol cm−2)

Γguest after release(μmol cm−2)

CB[8] binding efficiency (%)

102 (1st) 102 (2nd) 99 (3rd) 62 (1st) 63 (2nd) 64 (3rd) 13 7

44 51 52 37 39 41

93 82 76 40 39 37

a

From this, the binding efficiencies of CB[8] are calculated. Moreover, the incorporated amounts of 9-AnPy within the corresponding control experiments are shown.

Figure 3. Absorbance spectra of AnPy (left) and 9-AnPy (right) taken before (black) and after complexation with the binary hosts MV-CB[8] (red) or PMV-CB[8] (green), respectively. were converted into surface coverage values by calibration curves of both molecules (see Figures SI-2 and SI-3). The subsequent release of AnPy was performed by exposing the multilayer films to a solution of NaBH4, and absorbance was once again measured to identify the amount which was removed from the multilayer films. For control experiments using 9-AnPy, the reduction of the viologen side chain was not performed due to the fact that such isomer is excluded from the cavity of CB[8] and therefore cannot be released by the chemical treatment.

left). Both characteristics still indicate the inclusion, although they are no longer as pronounced as for the complex containing MV monomer. This might be due to the fact that the guest location within the PMV-CB[8] complex differs slightly in comparison to the monomeric complex. Another reason could be a slight inhibition effect on the inclusion of AnPy that was found earlier16 and attributed to the alkyl chain which is attached to the viologen moiety and therefore possibly marginally repels the guest molecule from the nanocontainer.16 We also considered the formation of micelles due to the introduced hydrophobicity of CB[8] after complexation with the cationic polyelectrolyte as a reason for the lesser pronounced flattening and shifting, respectively. However, since the distance between two CB[8] molecules on a single polyelectrolyte is rather large (see ratio of monomer units of the polyelectrolyte structure, Figure 2), and only a minor fraction of the polyelectrolyte charges are shielded by CB[8], we assume that the association of CB[8] is not sufficient to induce the formation of a hydrophobic micellar core in aqueous solution. When 9-AnPy was added to the binary hosts, no hypochromic shifts were observed, as expected (see Figure 3, right). In the case of MV-CB[8], the absorbances of both anthracene and pyridinium are only marginally decreased, while they remain constant in the case of PMV-CB[8]. This confirms again that the sterical demand of 9-AnPy sufficiently prevents an inclusion into MV-CB[8] or PMV-CB[8]. Nanocontainer Density in Polyelectrolyte Multilayer Films. We further addressed the nanocontainer density as well as its binding efficiency in multilayer films in dependence of the surface charge. Here, we used ultraviolet spectroscopy to quantify the imprinted amount of CB[8] in dip-coated PEM

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RESULTS AND DISCUSSION Host−Guest Properties of PMV-CB[8]. While we previously proved the principle of the novel SMILbL system,16 we are here interested in quantitative data concerning the efficiency of the binding. In order to quantify potential sterical hindrances of host−guest binding in multilayers, we studied the same host− guest system in solution for comparison. Therefore, the binary building block PMV-CB[8] is complexed in solution with the linear guest molecule AnPy. In addition, we compared it to the monomeric, binary complex MV-CB[8], consisting of monomeric methylviologen and CB[8]. When AnPy is offered to MV-CB[8], the inclusion into the nanocontainer is indicated by a small, but clearly observed hypochromical shift of the anthracene’s fine structure (ranging from ca. 320 to 400 nm) as well as by a significantly flattening41 (see Figure 3, left). Flattening is observed for the pyridinium absorbance in the range from 225 to 280 nm as well. Furthermore, a charge transfer occurred upon addition of AnPy toward MV-CB[8] (see Figure SI-16). When AnPy was added to the macromolecular host PMV-CB[8], a flattened shape and only a marginal shift of the maximum were observed for both the fine structure and the pyridinium moiety (see green line in Figure 3, 10737

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The decreasing absorbance of AnPy shown in Figure 5, top right, indicates the release of AnPy from the PEM as a result of the chemical reduction. As already observed for the preceding uptake process, the major amount of AnPy is removed within minutes, although it takes 1 h until the amount remains constant. As already mentioned, the same experiments were performed with a PEM terminated with a positively charged surface (see Figure 5, bottom). Herein, the uptake and release processes were again indicated by an increasing and decreasing absorbance, although the quantitative scale differs clearly with respect to the values obtained from the PEM terminated with a negatively surface charge. Guest uptake and release processes were also performed in subsequent cycles; Figure 6 summarizes the data of several uptake and release cycles of AnPy in dependence of the multilayer surface charge. The full spectra are given in Figures SI-6 and SI-7. The gust molecule surface coverage reached very similar values, indicating reversible behavior, although the coverage varies significantly depending on the charge of the terminating layer. Furthermore, the results of the control experiments using 9-AnPy are shown in Figure 6 (for spectra see Figures SI-8 and SI-9). Taking the initial uptake of AnPy into a negatively terminated PEM into account (Figure 6, left), one can observe that the uptake proceeds in an initial fast process within approximately 5−10 min. After that, the uptake continues as a slower process, finally resulting in a plateau at 102 μmol cm−2 (first uptake and release cycle, compare Table 1). We assume both the transport of the cationic molecule to the multilayer surface and the inclusion into CB[8] as the fast process, while the following decelerated uptake might represent the diffusion of AnPy into the deeper layers of the PEM. After saturation, the release experiment removed 58 μmol cm−2 (first uptake and release, compare Table 1 and Figure 6, left) of the incorporated AnPy. This value is in very good agreement with the surface coverage of CB[8] of 62 μmol cm−2 (see Table 1). Interpreting the remaining 44 μmol cm−2 as unspecifically bound, nonreleasable guest, these values indicate an excellent binding efficiency to 93% of the containers (Table 1). In addition, reversibility of the cycle is shown by multiple repetitions of the uptake and release experiments, leading again to high CB[8] binding efficiencies in the range of 76−93%. The fraction of AnPy that remains in the PEM after the release was completed (44−52 μM cm−2, see Table 1 and Figure 6, left) indeed stays constant throughout several binding and release cycles. This supports the above interpretation that it is not specifically bound by CB[8], but instead attracted in another unspecific fashion, e.g., by hydrophobic or electrostatic interactions. For verification, we employed 9-AnPy as the branched and therefore nonbinding isomer of AnPy to an identical PEM (see open squares in Figure 6, left). Herein, it is indicated that the incorporated amount of 9-AnPy, which is completely based on the above-mentioned unspecific interactions, is drastically lower than the remaining amount of AnPy (Table 1). The quantitative difference between the isomers might be due to the varying geometries of the molecules, since the linear structure of AnPy might be able to perform a stronger unspecific binding into the PEM as a result of the smaller hydrodynamic radius in comparison to the corresponding isomer. In addition, the kinetics of the uptake might play a role: As 9-AnPy has a nonlinear, more bulky structure, it is possible that its diffusive transport into the multilayer is kinetically hindered,

consisting of an initial cationic layer of PEI followed by eight adsorbed bilayers (PAA/PMV-CB[8]), resulting in PEMs having a positively charged surface. To create the corresponding PEM with a negatively charged surface, another PAA layer was adsorbed. Two broad absorption bands at λ = 223 nm (regime not shown) and λ = 263 nm indicate the formation of the PEM on the quartz substrates for both negatively and positively terminated multilayers (Figure 4). The band at λ = 263 nm

Figure 4. Absorbance of polyelectrolyte multilayer films consisting of eight PMV-CB[8] layers, either terminated cationically (red) or anionically (blue).

represents the absorbance of the viologen moieties of PMVCB[8]. Since the complex stoichiometry of CB[8] and viologen within the polyelectrolyte complex is 1:1, we could determine the surface coverage of the nanocontainer (ΓCB[8]) from the absorbance of the viologen side chain (APMV‑CB[8]; see Table 1, columns 2 and 3). The calculated values confirm both a good reproducibility and a coverage of incorporated CB[8] in the range of tens of μm cm−2, therefore revealing a considerable number of binding sites within the prepared polyelectrolyte multilayer films. Binding Efficiency of CB[8] in Dependence on PEM Surface Charge. After PEM preparation, the binding efficiency of AnPy was investigated, using 9-AnPy in a corresponding control experiment. As an interesting parameter, the influence of the multilayer surface charge on the binding of both molecules was monitored by employing them to a PEM either having a positively or negatively charged surface. Since an additional anionic layer does not provide CB[8], the amount of the nanocontainer is expected to be independent of the surface charge and thus constant for both PEM. This is indeed the case, as the data of the third column of Table 1 show. Since both molecules provide a significant absorbance in the range from 300 to 400 nm, it was possible for both AnPy (Figure 5) and 9-AnPy (Figures SI-4 and SI-5) to quantify the amounts incorporated into the multilayer film as well as studying the kinetics of the uptake and release processes. The spectra representing the uptake of AnPy into the negatively terminated PEM (Figure 5, top left) clearly show that saturation is reached after ca. 2 h, hence revealing a similar saturation time as already reported in our previous publication,16 although here the major amount of the molecule has initially been taken up within minutes. By reducing the viologen moiety into the corresponding radical, the attractive ion-dipole interaction between the side chain and AnPy is disabled, and therefore the latter is released from the nanocontainer. 10738

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Figure 5. UV−vis spectra of the first uptake (left) and release (right) cycle of AnPy into a negatively (top) or positively (bottom) terminated PEM.

Figure 6. Uptake and release cycles of AnPy (filled squares) into two single PEM with either a negative (blue data) or a positive (red data) surface charge. Incorporation of 9-AnPy (open symbols) into identically prepared CB[8]-containing multilayer films (in the case of 9-AnPy the reduction treatment was not performed).

interactions between the cationic 9-AnPy and PAA are one source of the unspecific uptake, among others. When AnPy is offered to a PEM with a positively charged surface (Figure 6, filled red squares), the kinetics is quite similar consisting of a rapid and a slow uptake on a minute to hour time scale, resulting in a plateau at 62 μmol cm−2. The subsequent reduction causes a release of 25 μmol cm−2; thus, the binding efficiency of CB[8] can be calculated to be 40%. Within two additional uptake and release cycles, the absolute values as well as the binding efficiency remain again constant (Table 1). Once more, there is a particular amount of AnPy remaining in the PEM as the result of additional unspecific binding, which is just slightly lower with respect to the PEM having a negatively charged surface (compare Figure 6 and Figure SI-11). From this, we conclude again that the additional PAA layer in the case of the

resulting in a lower unspecifically bound amount than expected for an electrostatically dominated equilibrium absorption. Generally, unspecific binding in this work is more strongly pronounced than in the previous paper, where films were crosslinked and continuously kept in solution.16 Our present procedure of intermittent drying for UV−vis measurements in dependence on uptake time, in combination with using noncross-linked films might cause such differences. Another effect can be found by taking the incorporated amounts of 9-AnPy in a PEM with either a negative or positive surface charge, respectively, into account (compare Figure 6 and Figure SI-10). For a negatively charged surface, the amount of 9-AnPy is slightly increased (Table 1), so we concluded that the additionally incorporated amount is related to the anionic nature of the outermost layer, and therefore electrostatic 10739

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Figure 7. Changes of the resonance frequency Δf during in situ formation of the multilayer films, yielding a multilayer film with either a positively (left) or negatively (right) charged surface. The colored background indicates the adsorption time of either the cationic (red) or the anionic (blue) polyelectrolyte. The initial PEI layer is shown in purple.

the cationic polyelectrolyte PMV was used without precomplexation of the nanocontainer (Figure SI-13). In this case ΔD was hardly increased during the cationic layer adsorption which confirms that the soft character of the PMV-CB[8] layers is clearly related to the presence of CB[8]. Kinetics of the Guest Molecule Uptake. The guest molecule uptake of AnPy was investigated concerning the uptake kinetics into as-prepared PEM terminated with either a cationic or anionic polyelectrolyte layer, yielding two QCM-D curves (Figure 8, previous PEM formation in Figure 7).

negatively terminated PEM is responsible for the slightly higher unspecific uptake of AnPy due to electrostatic interactions. Concerning the guest molecule recognition, it is obvious that the binding efficiency of CB[8] is strongly reduced just by changing the multilayer surface charge from negative to positive (Table 1). As an explanation for the observed quantitative difference, we assume the negatively charged multilayer surface to promote the transport of AnPy into the film and therefore leading to an almost complete coverage of the nanocontainer, while the positively charged surface on the other hand might act as an electrostatic barrier toward the cationic guest molecule and thus reduces both the transport into the PEM and, as a consequence, the inclusion into CB[8]. It is thus quite likely that the positively terminated film does not reach thermodynamic equilibrium concerning guest molecule uptake. In Situ Multilayer Film Formation Using QCM-D. Although ultraviolet spectroscopy is an excellent method for quantification, it requires a large effort to provide kinetic information on the guest molecule uptake and release into the film, and furthermore the achievable time resolution is limited (see Figure 6). Therefore, we assembled PEM, again either terminated with a negatively or positively charged surface, respectively, on a QCM-D quartz sensor in order to study the kinetics of the AnPy uptake behavior (see next section). The decrease of Δf with each polyelectrolyte adsorption demonstrates a regular formation of the PEM (see Figure 7 and Figure SI-15). The adsorbed mass of the cationic layer causes a significantly larger Δf decrease than that of the PAA layers. Thus, the adsorbed mass of the polycation considerably exceeds the mass of the anionic layer within each bilayer. Unfortunately, we were not able to reproduce the absolute mass coverage of the PEM precisely enough to obtain quantitative kinetic data from the QCM-D measurements. The film preparation is always quite regular (see Figure 7), but the final PEM vary strongly in their total absorbed mass, as indicated by a range of final values of Δf (550 Hz < Δf PEM < 700 Hz). Therefore, the observations monitored in QCM-D experiments are solely interpreted in a qualitative fashion. Additionally, an interesting feature of the PEM was observed in the dissipation data (see Figure SI-12). The adsorption of each cationic layer increases ΔD strongly, while the adsorption of PAA has only a negligible impact. From this we conclude that the bulky structure of the associated nanocontainer softens the cationic layers due to sterical reasons. This idea was confirmed by the preparation of a reference multilayer film where

Figure 8. Uptake kinetics of AnPy, obtained from QCM-D, into a multilayer film terminated either with a positively charged (top curve) or a negatively charged surface (bottom curve).

For the uptake of AnPy into a multilayer film having a negatively charged surface, the initially decreasing Δf indicates guest molecule incorporation into the PEM on a time scale of minutes. After that a slow Δf increase occurs over several hours, ending up in a plateau. According to the Sauerbrey equation, the increase of Δf indicates the release of mass from the sensor surface. An appropriate explanation of the observed Δf response can be found by combining the observations from both ultraviolet spectroscopy and QCM-D, respectively. As already shown in Figure 6, the PEM takes up the major amount of AnPy within ca. 10 min, leading to the conclusion that the initial mass uptake in QCM-D, seen in Figure 8, bottom curve, and occurring on the same time scale, represents the unspecific uptake as well as the inclusion into CB[8]. The following Δf increase indicates that material is released from the sensor surface. Furthermore, it must be noted that the 10740

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Langmuir

a general mass increase due to the AnPy uptake could be detected for a PEM having a negatively charged surface, it was not visible when the surface charge was changed to positive. For the latter we assign the absence of the mass uptake as the result to a decreased amount of incorporated AnPy, which was previously found by ultraviolet spectroscopy. Besides the unspecific uptake, quartz crystal microbalance revealed another kinetic process during the guest molecule uptake, showing that a deswelling, i.e., a release of hydration water, occurs when the guest molecule penetrates the multilayer film.

amount of released mass in fact exceeds the mass which was previously incorporated. With respect to the prolonged time scale of the mass release, we assume that it cannot be caused by AnPy since its incorporated amount remained constant (compare Figure 6). In addition, the simultaneously detected dissipation change ΔD does not show any drastic changes and therefore excludes a potential decomposition of the PEM caused by the guest (see Figure SI-14). Taking these facts into account, the only component remaining as a candidate for mass release is hydration water of the PEM. This is why we assign the slow Δf increase to be a deswelling of the PEM as a consequence of the guest molecule uptake. Within the deswelling, the PEM is compressed and therefore hydration water is released from the interior, detected as increasing Δf response. As a potential reason, we postulate an electrostatic disequilibrium, i.e., an excess of negative charges of the as-prepared PEM. Then, upon guest uptake, the compression of the PEM is a result of the introduced cationic charges of the incorporated AnPy. This mechanism is similar to the strong swelling and deswelling observed in a PEM-based imprinting system with electrostatic interactions between the guest and the binding site.15 An initial mass uptake is no longer detected for the uptake of AnPy into a PEM bearing a positively charged surface (see Figure 8, top curve). Instead, the immediate mass release indicates that the deswelling is instantaneously initiated when AnPy penetrates the PEM. The absence of an initial mass uptake can be reasonably explained since the incorporated amount of AnPy into the PEM was strongly reduced due to the change of the multilayer surface charge from negative to positive (see Figure 6). We assume that the deswelling is in general instantaneously initiated by AnPy, but in the case of negatively charged multilayer surface it was superimposed by the enhanced mass of AnPy which bound into CB[8]. As a consequence, the release of hydration water from the PEM with a negatively charged surface was not visible until the multilayer film has been saturated with AnPy after approximately 10 min (see Figure 6, right).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b02806. Detailed UV−vis spectra of cyclic guest molecule uptake and release and further supporting spectra and QCM-D traces (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(M.S.) E-mail [email protected], Ph +492518323419, Fax +49-2518329138. Present Address

J.Z.: Ningbo Institute of Material Technology and Engineering, Chinese Academy of Science, 1219 Zhongguan West Road, Ningbo 315201, P. R. China. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are thankful to the Deutsche Forschungsgemeinschaft (DFG, Germany) and the National Science Foundation of China (NSFC, China) for funding this work as a part of the Sino-German collaborative research center SFB TRR61, project B11.

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CONCLUSIONS In this work we assembled cucurbit[8]uril as a nanocontainer into polyelectrolyte multilayer films and used it successfully for the recognition and inclusion of a guest molecule. By employing ultraviolet spectroscopy, we determined quantitative characteristics such as the imprinting density as well as the binding efficiency of cucurbit[8]uril in dependence of the multilayer film surface charge. We were able to prepare multilayer films with a quite high content of CB[8] of 62 μmol cm−2. Further investigations indicated that the binding of the guest molecule into the nanocontainer quantitatively depends on the surface charge of the multilayer film, leading to an excellent binding efficiency in the case of a negatively charged multilayer film surface (76−93%). However, the binding efficiency was drastically decreased when the multilayer film surface was positively charged (37−40%), leading to the conclusion that a cationic outermost layer has a repellent effect toward the cationic guest molecule, while an anionic outermost layer promotes its uptake into the film and therefore supports the binding into the nanocontainer. The binding process is accompanied by an additional unspecific uptake of AnPy into the PEM due to additional attractive interactions. Dissipative quartz crystal microbalance was employed to investigate the guest molecule uptake kinetics in situ. Again, a dependence on the multilayer surface charge was found. While

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Article

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DOI: 10.1021/acs.langmuir.5b02806 Langmuir 2015, 31, 10734−10742