Chem. Mater. 2005, 17, 1007-1016
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Molecularly Imprinted Polymer Films with Binding Properties Enhanced by the Reaction-Induced Phase Separation of a Sacrificial Polymeric Porogen Ronald H. Schmidt† and Karsten Haupt*,‡ Lund UniVersity, Department of Pure and Applied Biochemistry, P.O. Box 124, 22100 Lund, Sweden, and Compie` gne UniVersity of Technology, UMR CNRS 6022, BP 20529, 60205 Compie` gne Cedex, France ReceiVed September 16, 2004. ReVised Manuscript ReceiVed December 14, 2004
Molecularly imprinted polymers (MIPs) are synthetic materials that mimic the behavior of natural antibodies while exhibiting far greater stability than their natural counterparts. Although potentially very useful as recognition elements in chemical sensors, a major obstacle to their widespread application has been the difficulty in preparing MIPs in the thin-film format that is necessary for coupling them to interrogative transducers. This paper offers a solution to this problem by presenting a straightforward approach to the in situ synthesis of MIP films with good control of thickness and porosity. Spin coating is used to spread a pre-polymerization mixture, which is polymerized in situ with UV light. A key aspect of this process is the reaction-induced phase separation between the rapidly polymerizing acrylate monomers and a sacrificial linear polymer porogen. We studied the degree of phase separation and the ability of the films to rebind the target analyte as functions of the concentration and molecular weight of the polymer porogen, the volatility of the nonreactive solvent, and the spin rate used during polymerization. The current focus is on producing films with thicknesses ranging from approximately 1 micron to 10s of microns; however, the technique can easily be adapted to prepare films as thin as 10s of nanometers, enabling their use as recognition elements for a wide variety of chemical sensing platforms.
Introduction The synthesis of materials with molecular recognition properties has become a topic of extreme technological and scientific interest. One of the most popular strategies for preparing synthetic receptors is molecular imprinting.1 This technique involves the copolymerization of functional and cross-linking monomers in the presence of a molecular template. A template is chosen that is similar or identical to the final analyte, and functional monomers are selected that can complex with the template via hydrogen bonding, electrostatic forces, van der Waals forces, or hydrophobic interactions.1 Alternatively, a template-monomer adduct is preformed via reversible covalent bonds.2 A large excess of the cross-linker ensures that the functional monomers are held rigidly in place, so that following polymerization and the subsequent extraction of the template, the molecularly imprinted polymer (MIP) retains a “molecular memory” of the template. The affinity and selectivity of binding sites that are created by this process often rival the properties of natural antibodies, enzymes, and cells. Compared to biological entities, however, MIPs possess exceptional chemical and thermal stability. Molecular imprinting also enables the preparation of receptors for target species for which no natural antibody or enzyme is known to exist. These advantages render MIPs very desirable for use as recognition elements in chemical * E-mail:
[email protected]. † Lund University. ‡ Compie ` gne University of Technology. E-mail:
[email protected].
(1) Sellergren, B. Angew. Chem., Int. Ed. 2000, 39, 1031-1039. (2) Wulf, G. Angew. Chem., Int. Ed. 1995, 34, 1812-1832.
sensors, particularly when operating in harsh environments.3 Unfortunately, the full potential of MIP-based sensors has not yet been realized, partly because of the difficulty of producing MIPs in the film format that is required for most mechanisms of transduction. An early strategy for constructing MIP-based sensors involved coating the surface of the transducer with particles that were polymerized beforehand.4,5 Although conceptually simple, such methods suffer from several serious drawbacks, including poor control of the coating thickness, regions of the substrate that are left bare, and a tendency of the particles to desorb from the surface of the transducer. Frequently, additives (e.g., PVC) are used to create a matrix that helps the particles adhere and form a cohesive layer,5 but such additives may block access to the binding cavities, thereby interfering with the rebinding of the analyte, and the composite films are unstable in solvents that are capable of dissolving the matrix. Most researchers in the field have therefore attempted to develop strategies for preparing MIP films in situ. One technique involves “sandwiching” a traditional pre-polymerization mixture between the transducer and a second, sacrificial substrate that is removed after polymerization.6 The thickness of films produced in this manner can be varied (3) Haupt, K.; Mosbach, K. Chem. ReV. 2000, 100, 2495-2504. (4) Kro¨ger, S.; Turner, A. P. F.; Mosbach, K.; Haupt, K. Anal. Chem. 1999, 71, 3698-3702. (5) Liang, C.; Peng, H.; Bao, X.; Nie, L.; Yao, S. Analyst 1999, 124, 1781-1785. (6) Jakusch, M.; Janotta, M.; Mizaikoff, B.; Mosbach, K.; Haupt, K. Anal. Chem. 1999, 71, 4786-4791.
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over the range of a few microns by adjusting the pressure that is applied to the sandwich. Disadvantages include the limited range of thicknesses obtained, the inhomogeneity of the films, and the difficulty in coating large areas. Much thinner films can be prepared using electrochemical polymerization techniques,7,8 but these methods require conductive substrates, and the technique is only compatible with a small variety of functional groups, compared to the wide assortment of functional monomers that have been employed by more traditional imprinting protocols which are based on freeradical polymerization. Others have attempted to adapt surface initiation techniques.9,10 In principle, surface initiation enables the synthesis of thin films (h < 200 nm) that are covalently bound to the substrate. In practice, however, the control of thickness and homogeneity that has been demonstrated for the preparation of linear films of polystyrene11,12 has, to our knowledge, not been reproduced for MIP films; most applications of the technique have involved preparing coatings for spherical silica beads that are more useful for chromatography or solid-phase extraction.10 These difficulties have led researchers to question whether it is possible to borrow standard techniques from the microelectronics industry that are used for preparing photoresist, dielectric, and antireflection coatings. In particular, spin coating has generated considerable interest because of the good control of thickness and homogeneity that the technique enables.13 Spin coating generally involves dissolving or dispersing the material to be deposited in a solvent, dispensing the mixture onto a flat substrate, and spinning the substrate at high speeds until all of the solvent has evaporated, leaving behind a thin coating of the solute. The technique had previously enjoyed little success with imprinters, however, which is due in part to the (necessarily) highly cross-linked structures of MIPs that render them impossible to dissolve. Liang et al. have attempted to overcome this problem by dispersing MIP particles in a solution of poly(vinyl chloride) (PVC) dissolved in tetrahydrofuran.5 This apparently yielded satisfactory results for the coating of a bulk acoustic wave sensor that functioned under ideal, research lab conditions; however, the composite films that are produced in such a manner suffer from serious shortcomings, as previously described. We recently communicated a versatile and robust technique that, for the first time, enables the in situ synthesis of spin-coated MIP films.14 Key aspects of the process include the use of low volatility components and a sacrificial linear polymer dissolved in the pre-polymerization mixture. Rigor(7) Malitesta, C.; Losito, I.; Zambonin, P. G. Anal. Chem. 1999, 71, 13661370. (8) Panasyuk, T. L.; Mirsky, V. M.; Piletsky, S. A.; Wolfbeis, O. S. Anal. Chem. 1999, 71, 4609-4613. (9) Schweitz, L. Anal. Chem. 2002, 74, 1192-1196. (10) Ru¨ckert, B.; Hall, A.; Sellergren, B. J. Mater. Chem. 2002, 12, 22752280. (11) Huang, W.; Skanth, G.; Baker, G.; Bruening, M. Langmuir 2001, 17, 1731-1736. (12) Schmidt, R.; Zhao, T.; Green, J.; Dyer, D. J. Langmuir 2002, 18, 1281-1287. (13) Hall, D. B.; Underhill, H. P.; Torkelson, J. M. Polym. Eng. Sci. 1998, 38, 2039. (14) Schmidt, R. H.; Mosbach, K.; Haupt, K. AdV. Mater. 2004, 16, 719722.
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ous evaluation of the binding properties required the development of novel characterization techniques. The current paper presents the results of a thorough investigation of the reaction-induced phase separation that leads to the enhanced binding properties of these films. The relationships between the porogen composition, film morphology, and binding properties are examined, and strategies are presented for controlling the film thickness. As a model template, we chose the commonly prescribed β-blocking drug, S-propranolol. The presence of a chiral center enabled us to demonstrate that the selectivity of the MIP films produced by this method is comparable to that which is observed in traditional bulk MIPs. Furthermore, the commercial availability of radiolabeled S-propranolol enabled the straightforward characterization of the binding properties by autoradiography. Experimental Section Materials and Equipment. Trimethylolpropane trimethacrylate (TRIM), poly(vinyl acetate) (PVAc), and 2,2-dimethoxy-2-phenylacetophenone (DMPA) were purchased from Aldrich. Methacrylic acid (MAA) was obtained from Merck. R- and S-propranolol hydrochloride, diglyme, and triglyme were obtained from Fluka. 3H-labeled S-propranolol was purchased from Perkin-Elmer. N-type, phosphorus-doped, 4-in. silicon (100) wafers were obtained from Pi-Kem Ltd. The spin coater (Specialty Coating Systems, Inc., Model P6700) was modified with a cover that enabled us to purge the bowl with argon prior to beginning the spin cycle and photolysis. To permit the penetration of UV light, the cover had a window made from plastic wrap film, which transmitted 70% of incident 254-nm light, as determined with a spectrophotometer. Synthesis. The silicon wafers were cut into smaller pieces (approximately 3 × 3 cm squares) and cleaned by soaking them for at least 15 min in a 1:1 solution of concentrated H2O2/NH4OH at ca. 70 °C. The wafers were then rinsed with deionized water and methanol, dried with a jet of compressed air, and immersed overnight in a solution that contained 2% (v/v) 3-(trimethoxysilyl) propyl methacrylate in toluene. The silanized wafers were washed in toluene, rinsed with methanol, and blown dry with a jet of compressed air. The reaction mixture contained a 1:10:35 molar ratio of template, functional monomer, and cross-linker, respectively. Except where otherwise noted, the volume ratio between the porogen and the monomers (i.e., MAA and TRIM) was kept at 4:3. The amount of initiator that was used was 1.7%, relative to the number of moles of polymerizable groups. Preparation of the reaction mixture involved dissolving an appropriate amount of DMPA in TRIM (22.3 µL/mg of DMPA), MAA (1.70 µL/mg of DMPA), and the porogenic mixture (diglyme, triglyme, or a solution of PVAc in one of these solvents; 32 µL/mg of DMPA). S-Propranolol was dissolved in this solution (108.5 µL/mg of S-propranolol), and the mixture was sparged with argon for 5 min at 0 °C. The prepolymerization mixture was pipetted onto the polished side of a silanized wafer, and the substrate was then placed onto the chuck of the spin coater. After purging the bowl for 30 s with argon, a 30-s spin cycle was commenced while simultaneously illuminating the contents of the bowl with an 8-W, 254-nm, low-pressure mercury lamp (Sterilair, model UVC-9; lamp-to-sample distance approximately 5 cm). After the spin cycle completed, the purge flow was stopped, the sample was left in the bowl, and the photolysis was continued for another 10 min. Each sample was soaked overnight in toluene, followed by washing for 6 h in a
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Table 1. Vapor Pressures of the Solvents and Monomers Used in This Study and of Other Commonly Used Imprinting Solvents solvent or monomer chloroform acetonitrile toluene diglyme triglyme MAA TRIM
vapor pressure (Torr) at 25 °Ca 200 170 28 3.2 0.2 1.2 2.5 × 10-7
a
Obtained from SciFinder Scholar and calculated using Advanced Chemistry Development (ACD) Software Solaris v4.67.
mixture of 1:4 acetic acid/methanol, rinsing with methanol, and drying under vacuum. Characterization. The binding properties of the films were evaluated by cutting a small piece (typically 3 × 3 mm) from each sample and incubating it overnight in 300 µL of a solution that contained 30 nM 3H labeled S-propranolol and 0.5% acetic acid in toluene. The samples were then rinsed for 20 s in 1 mL of toluene and allowed to dry (initially at ambient conditions and then under vacuum). Incubated samples were exposed overnight to a 3H-sensitive storage phosphor screen (Amersham Biosciences), which was then scanned by a Storm Phosphoimager. Molecular Dynamics ImageQuant v5.0 software was used to evaluate the autoradiography images and to determine the concentration of radioligand in each of the films. Competitive binding was performed as above, using various concentrations of unlabeled R- and S-propranolol. Film morphology was characterized using a Digital Instruments MultiMode AFM operated in contact mode, and the thicknesses of the films were determined using a Dektak 3030 profilometer.
Results and Discussion Formulation and Binding Properties of Spin-Coated MIP Films. Our initial attempts to cast MIP films employed reaction mixtures with compositions similar to those that are commonly used to prepare MIPs in the bulk monolith format: MAA and TRIM were used as the functional and cross-linking monomers, toluene was chosen as the porogenic solvent, and azobisisobutyronitrile (AIBN) was used as the free-radical initiator. Very long reaction times were needed to polymerize the monomers, however, and the toluene evaporated very early during the spin cycle, resulting in nonporous films with virtually no binding capacity for radiolabeled propranolol. The problem of rapid solvent evaporation was solved by replacing toluene with the less volatile solvent diglyme (refer to Table 1 for vapor pressures), and much faster polymerization rates were achieved by replacing the AIBN with DMPA. The resulting films were nontacky after relatively short photolysis times (10 min), and they exhibited a smooth, glossy, and uniform appearance. Surprisingly, the binding properties of these films were only marginally improved compared to the films made with toluene. Figure 1A shows an autoradiography image of a portion of one of these films after incubating the sample in a solution that contained 3H-labeled S-propranolol. The contrast between the background and the region in the center that corresponds to the film was much smaller than expected, indicating low binding of the radioligand. To understand this result, we characterized the morphology of the film with an AFM. The topography image in Figure 1B reveals small and
Figure 1. (A) 8 × 8 mm autoradiography image of a film prepared with diglyme at 2000 rpm and exposed to 3H-labeled S-propranolol. Darker colors represent regions of higher concentrations of beta radiation. (B) A 5 × 5 µm AFM image of the topography. The Z-scale is 10 nm, and darker and lighter colors correspond to regions of lower and higher topography, respectively.
apparently random fluctuations in height, and the magnitude of these fluctuations is described by a root-mean-square (RMS) roughness of 0.61 nm, which is comparable to the roughness observed in spin-cast films of linear homopolymers. This relatively featureless morphology is very different from the well-defined pore structure observed in MIPs that were prepared as bulk monoliths. A mean film thickness of 1.0 µm was determined by profilometry of scratches produced at three different regions of the same film, and a small relative standard deviation (5% RSD) indicates that the coating thickness is uniform. The reproducibility of the procedure was confirmed by preparing a total of three films under nominally identical conditions, resulting in a mean thickness of 0.95 µm with a 7% RSD. Gravimetric measurements of the substrates before and after casting and washing the films resulted in a mean mass per unit area of 1.77 g/cm2. A large relative standard deviation among the three films (12%) is more likely because of uncertainty in the weight measurements rather than to differences between the films. Calculation of the density of the films is straightforward: F)
m/a h
(1)
where F is the density, m/a is the mass per unit area, and h is the thickness. Using the mean values of m/a and h reported above results in an estimated density of 1.87 g/cm3.15 The above results suggest that the films prepared with a porogen consisting of pure diglyme are nonporous. The formation of a porous MIP involves the phase separation of the growing cross-linked network from the porogen. Although bulk MIPs that were prepared with diglyme were highly porous (data not shown), we postulated that the rapid polymerization kinetics prevented phase separation in the present case. One way to accelerate the kinetics of phase separation is to increase its thermodynamic driving force, which we attempted to accomplish by dissolving linear polymers in the pre-polymerization mixture. PVAc was chosen because it is soluble in the initial reaction mixture (15) While this estimate is surprisingly large, the remaining films considered in this study were synthesized and characterized in a similar manner; thus, the relative systematic errors in the determination of their densities are likely to be uniform. Ultimately, we are interested in the differences between the densities of these remaining films, relative to the density of the films prepared with the diglyme porogen. Therefore, we expect that the pore volumes that we calculate from these density measurements are reasonably accurate.
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Figure 3. Competitive binding study, demonstrating the chiral selectivity of a film prepared in the presence of 5% PVAc140 at 2000 rpm. The initial concentration of the radioligand (3H-labeled S-propranolol) was fixed, but different concentrations of unlabeled R- and S-competitor ligands were used.
Figure 2. Top: Autoradiography images of films prepared at 2000 rpm with different concentrations (wt %, relative to the pure diglyme)16 and molecular weights of PVAc. Bottom: Relative concentration of bound radioligand vs PVAc concentration. The relative signal corresponding to the films prepared with 0% PVAc has a value of unity.
and it does not possess functional groups that are likely to interfere with the formation of the template-monomer complex. Three different distributions of molecular weight were considered for the polymer porogen, with weightaverage molecular weights (M h w) of 83 × 103, 140 × 103, 3 and 500 × 10 g/mole. Hereafter, these will be designated as PVAc83, PVAc140, and PVAc500, respectively. Figure 2 shows autoradiography results from a series of experiments in which the concentration and the molecular weight of the PVAc additive were each systematically varied.16 After inspecting the raw data at the top of the figure, it is immediately apparent that the films prepared in the presence of PVAc exhibited much higher autoradiography signals than those prepared without PVAc. We quantified these effects using image analysis software, and the results from this analysis are plotted at the bottom of Figure 2. The vertical axis represents the binding signal of each film, relative to the signal obtained for the samples prepared without PVAc. For PVAc83, the concentration of bound ligand increased monotonically, with a 40-fold increase in the signal observed at 10% (wt %, relative to the pure diglyme). The addition of PVAc140 and PVAc500 resulted in a similar enhancement of the binding properties, but maximum signals in these cases were observed at 7.5% PVAc, with apparent decreases at 10% PVAc. Understanding these results requires a rigorous characterization of the film morphology, which we will present later in this paper. The selectivity of the films was evaluated with a competitive binding study, and as a representative example, we chose the film that had been prepared with 5% PVAc140. The data (16) The concentration of PVAc is defined throughout this paper as the nominal volume percentage relative to the pure solvent. However, prior to polymerization the porogen is diluted with monomers, so that a nominal concentration of 10% PVAc actually represents a diluted concentration of 4.3%. This discrepancy decreases during the polymerization, however, so that the final true PVAc concentration is the same as the initial, nominal concentration.
from this experiment are shown in Figure 3. Samples that contained low concentrations of competitor ligand exhibited high autoradiography signals. As the concentration of either of the unlabeled ligands was increased, however, the binding of the radioligand was inhibited, and the resulting drop in the signal occurred more rapidly for the unlabeled S-propranolol competitor than for the R-propranolol competitor. We estimated a cross-reactivity of 10%, which demonstrates the high enantioselectivity of the films prepared in this manner.17 Thickness and Density of Porous Films. Profilometry and gravimetry were used to characterize the thicknesses of the films synthesized in the presence of PVAc, and the results are shown in Figure 4. The mass per unit area (m/a) increased approximately linearly with the PVAc concentration, while a more rapid increase was observed in the thickness measurements. This observation leads us to conclude that the density of the films, as described by eq 1, decreases with increasing PVAc content. By comparing the density of a porous film to that of a nonporous film (synthesized in the absence of PVAc), we can calculate the fractional volume occupied by pores: Vpore ) 1 -
FPVAc F0
(2)
where F0 and FPVAc represent the densities of nonporous and porous MIP films, respectively. As an estimate of F0, we can use the mean value reported above (1.87 g/cm3) for the films prepared in the absence of PVAc.15 Experimental uncertainty in the m/a measurements makes it difficult to accurately determine the pore volume at low PVAc concentrations. However, at a polymer porogen concentration of 10%, we estimated Vpore values of 0.5 for PVAc83 and 0.6 for PVAc140 and PVAc500. It is important to compare the thicknesses of the films to the penetration depth of the beta particles emitted by tritium. Beta particles having the mean (most characteristic) energy of 5.7 eV can travel approximately 400 nm through an organic film that has a density of 1 g/cm3. Because of the (17) Cross-reactivity is defined as CR ) (IC50S/IC50R) × 100, where IC50S and IC50R are the concentrations of S- and R-ligands that inhibit 50% of the binding between the MIP and the radioligand.
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We will next examine the relationship between the molecular weight of the PVAc and the morphology of the films. Consider, for example, the film that had been prepared with 2% PVAc83 (Figure 5B). The morphology is characterized by smaller, more discrete pores compared to the relatively large and partially coalesced holes in the film that had been prepared with 2% PVAc140. Contrast inversion occurred in the film prepared with 3% PVAc83, but the high topography domains are smaller and thinner in comparison with the islands in the 3% PVAc140 film. Further increasing the concentration of PVAc83 eventually led to a grainy structure, but it is clear that higher concentrations of linear polymer were required to reach this morphology.
Figure 4. (A) Mass per area measurements plotted as a function of PVAc concentration for films prepared at 2000 rpm. (B) Thickness measurements vs PVAc concentration.
rapid energy loss for beta particles originating beneath the polymer/air interface, however, the maximum film depth that contributes to the autoradiography signal is probably significantly less than this maximum penetration depth. We therefore conclude that differences in the autoradiography signal are due to intrinsic differences between the morphologies of the films and not to differences in their thickness. Morphology and Reaction-Induced Phase Separation. An understanding of the mechanism of pore formation requires us to examine the morphology of the films. Figure 5 shows representative AFM topography images of films that were prepared with different porogen compositions. We will first consider those films that were prepared in the presence of PVAc140. At a concentration of 1%, the morphology consists of discrete, round holes that are difficult to see in the 50 × 50 µm scan (Figure 5G) but are more visible in a 15 × 15 µm scan (Figure 5S). Increasing the concentration to 2% resulted in much larger pores, many of which coalesced into irregularly shaped holes. In some cases (refer to the boxed-in region in Figure 5T), these holes have coalesced around islands of high topography, but for the most part, the higher topography regions remain continuous. Further increasing the PVAc140 content to 3% resulted in a phase inversion (or contrast inversion), such that the low topography features now comprise the continuous phase, and the higher topographical features are present as discrete islands that appear fairly uniform in size and shape. Increasing the resolution of the scan and changing the color scale (Figure 5U) reveals small spherical grains scattered throughout the continuous, low topography domain. With further increases in the PVAc140 concentration, these spherical grains eventually dominate the morphology.
Increasing M h w to 5 × 105 g/mole led to interesting changes in the morphology. Consider the film prepared with 2% PVAc500, which at first glance appears to be bicontinuous. Closer inspection, however, reveals that the high topography features are actually discontinuous islands that are dispersed throughout the continuous, low topography background. Thus, the contrast inversion that first appeared at 3% PVAc for the two lower molecular weights has shifted to a lower concentration when PVAc500 was used. A higher resolution image of the 2% PVAc500 film (Figure 5V) reveals that the islands contain small, discrete pores that resemble those seen at lower PVAc concentrations. Similar pores were not visible at higher concentrations of PVAc500. Another interesting feature is the anisotropy visible in Figure 5P. The large grains present in this film are stretched along an axis parallel to the spin direction, which is indicated in the figure by the white arrow. The high viscosity of the reaction mixture probably contributed to this anisotropy, and similar features were not seen in the films that were prepared with the lower molecular weights of PVAc. We now turn to a quantitative evaluation of the various morphologies described above. We begin with measurements of the RMS roughness of the films, which are plotted as a function of PVAc concentration in Figure 6. Ignoring random error, we see a monotonic increase in the roughness as a function of PVAc content for the lower two molecular weights. By contrast, the films prepared with PVAc500 displayed a maximum roughness at a concentration of 7.5%, and the film prepared with 10% PVAc500 was much less rough than films synthesized with similar concentrations of PVAc83 and PVAc140. The interpretation of these roughness measurements is not straightforward, and it is arguably more interesting to examine the lateral length scale of the fluctuations in height. This length scale is determined from a Fourier analysis of topography images, and representative two-dimensional power spectra are shown at the top of Figure 7. Inspection of the spectra reveals isotropic rings for all of the films prepared with e4% PVAc83 and PVAc140, as well as for the films prepared at low concentrations of PVAc500. However, the anisotropy observed in the film prepared with 4% PVAc500 (Figure 5P) is also evident in its power spectrum. Despite this irregularity, all of the 2D spectra were converted into isotropic (i.e., angularly averaged) power spectra and integrated to obtain a mean length scale, λh, which is approximately equal to the correlation length of the features. The results from this analysis are plotted as a
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Figure 5. A-R: 50 × 50 µm topography images of films prepared with different porogen compositions at 2000 rpm. The number in the bottom right-hand corner of each image describes the Z-range of the color scale. The arrow in image P describes the spin direction. S-V: 15 × 15 µm topography images with a 250-nm color scale. Images S, T, and U correspond to the films prepared with 1, 2, and 3% PVAc140, respectively. Image V corresponds to the film prepared with 2% PVAc500.
function of PVAc concentration at the bottom of Figure 7. Initially, λh is approximately proportional to the concentration of PVAc, but eventually a maximum value is reached, and
the position of this maximum coincides with the transition from discrete domains to the (nonspatially correlated) grainy features.
Molecularly Imprinted Polymer Films
Figure 6. Root-mean-square (RMS) roughness vs PVAc concentration for the films shown in Figure 5.
Figure 7. Top: 2D power spectra of representative images from Figure 5. Bottom: Characteristic length scale vs PVAc concentration.
We will now consider which of the two principal mechanisms of phase separation (nucleation and growth or spinodal decomposition) occurs between the porogen and the growing MIP network. In general, which mechanism dominates is usually dependent on whether the system is within the metastable region (NG) or the unstable region (SD) of the phase diagram, and this in turn is determined by the
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quench depth.18 In SD, the early and intermediate stages of separation are often characterized by bicontinuous patterns that resemble the (quasi)bicontinuous morphology shown in Figure 5N. The remaining films did not exhibit such patterns; however, the short-range order that we observed at low and intermediate PVAc concentrations is more consistent with SD than with NG, since the latter mechanism produces nucleation sites that are randomly distributed.19 The increase in correlation length with the concentration or molecular weight of the polymer porogen is analogous to the coarsening process in a film that undergoes thermally induced spinodal decomposition.20 However, films that were prepared with high concentrations of PVAc do not exhibit short-range order; instead, the morphology seems to result from the separation of droplets of oligomers and monomers that subsequently polymerized into an agglomerated film. Similar morphologies have been seen in advanced stages of separation for a thermally quenched blend that exhibited a bicontinuous pattern at earlier stages.21 We therefore conclude that increasing the concentration or molecular weight of the PVAc porogen has the effect of accelerating the spinodal decomposition that leads to the more porous morphologies. For a thermal quench of an ideal binary blend of polymers, it is fairly easy to apply Flory-Huggins theory to construct binodal and spinodal curves that separate the stable, metastable, and unstable regions of the phase diagram.18 Modeling reaction-induced phase separation22-24 is less straightforward, since at a fixed temperature the quench depth increases during the polymerization. Perhaps the simplest case involves a condensation polymerization in the presence of nonreactive linear polymers. Ishii and Ryan studied a system containing a linear polymer dissolved in epoxy resin.23 The gradual, continuous increase in the molecular weight of the resin enabled the authors to assume pseudobinary behavior. This assumption involved setting the initial degree of polymerization of the “reactive solvent” to unity and allowing it to increase with time, resulting in binodal and spinodal curves that shifted as the degree of polymerization increased during the reaction. By contrast, the free-radical (step-growth) polymerization that we are considering is characterized by a discontinuous distribution of molecular weight (i.e., high molecular weight polymer dispersed in the monomers), and the system is further complicated by the presence of crosslinks, so that a ternary phase diagram becomes necessary to describe the phase behavior. Liskova and Berghmans demonstrated this approach by constructing empirical ternary phase diagrams for a reacting system that contained styrene, linear polystyrene, and paraffin wax.24 Controlling Film Thickness while Maintaining a High Binding Capacity. Most chemical sensor applications require (18) Flory, P. J. Principles of Polymer Chemistry; Cornell University Press: Ithaca, NY, 1953. (19) Tanaka, H.; Araki, T. Phys. ReV. Lett. 1998, 81. (20) Siggia, E. D. Phys. ReV. A 1979, 20, 595-605. (21) Heier, J.; Kramer, E. J.; Revesz, P.; Battistig, G.; Bates, F. S. Macromolecules 1999, 32, 3758-3765. (22) Nephew, J. B.; Nihei, T. C.; Carter, S. A. Phys. ReV. Lett. 1998, 80, 3276-3279. (23) Ishii, Y.; Ryan, A. J. Macromolecules 2000, 33, 158-166. (24) Liskova, A.; Berghmans, H. J. Appl. Polym. Sci. 2004, 91, 22342243.
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diglyme with the less volatile solvent triglyme. Thickness and m/a measurements of MIP films formulated with triglyme are plotted in Figure 8B, and both parameters vary by a factor of approximately 5 over the range of spin rates that we are considering. Also, the density of the films appears independent of the spin rate. Autoradiography results are shown in Figure 8C. At 1000 rpm, films produced with diglyme/PVAc140 and triglyme/ PVAc140 exhibited similar autoradiography signals. The two data series diverge at higher spin rates, however, with a decrease in the signal observed for the diglyme-based formulation and a relatively constant signal when triglyme was used as the solvent. These trends are consistent with the density measurements shown in Figure 8A and 8B. The increase in density observed for the diglyme-based formulation at high spin rates implies (via eq 2) a decrease in the volume fraction of the pores. In the triglyme-based formulation, the constant density implies a pore volume that is independent of the spin rate, which is consistent with the constant autoradiography signal. For more traditional spin-coating processes that involve the deposition of nonreactive solutes dissolved in volatile solvents, it is possible to predict the rate of film thinning (dh/dt) for a given spin rate (ω), solution viscosity (η), and evaporation rate (e):25 dh -2Fω2h3 ) -e dt 3η
Figure 8. A, B: Mass per area and thickness measurements are plotted on the left axis as a function of spin rate, and the calculated density is plotted on the right axis. C: Autoradiography signal vs spin rate.
an optimum thickness of the selective adsorption medium to maximize the signal detected by the transducer. Toward this end, we have considered different methods for controlling the thickness of the films. For a fixed composition of the pre-polymerization mixture, varying the spin rate is the most straightforward strategy. The first series of films that we will consider were formulated with diglyme, which is the same solvent that was used in the above results, and all films considered in this section were prepared with 10% PVAc140. Mass per area and thickness measurements are plotted as a function of spin rate in Figure 8A. Both measurements decrease monotonically with spin rate, but the thickness decreased more rapidly than m/a. Using eq 1, we calculated a film density that increased from approximately 0.8 g/cm3 at 1000 rpm to 1.4 g/cm3 at 8000 rpm. These results suggest that the more volatile components of the prepolymerization mixture may have evaporated at the higher spin rates. Inspection of the entries in Table 1 reveals that diglyme has a much higher vapor pressure than MAA or TRIM, implying that the solvent would be expected to evaporate faster than either monomer. Thus, we replaced
(3)
The creation of nonporous morphologies when toluene was used (or when diglyme was used at high spin rates) highlights the importance of using components with low vapor pressures. Thus, the evaporation rate can ideally be ignored for the current system. Nevertheless, the solution of eq 3 is not trivial because of the rapidly increasing viscosity during polymerization, which eventually leads to the gelation of the film and the prevention of further thinning. These considerations suggest a straightforward method for characterizing the polymerization kinetics. When the spin cycle and photolysis are initiated simultaneously, one would expect to see a time-dependent thickness for short spin cycles and a constant thickness for long spin cycles. That is, a plot of thickness versus duration of the spin cycle, tspin, would exhibit a cusp at the gel point, tgel, with an initial decrease in thickness for tspin < tgel, and constant thickness for tspin g tgel. We varied the spin time between 5 and 30 s and found a constant thickness, which suggests that tgel e 5 s. This rapid polymerization is in dramatic contrast with the 1-3 h typically required to reach the cloud point (which indicates the onset of phase separation) for MIPs prepared in the bulk monolith format. Correlating Morphology with Binding Properties. We now return to AFM imaging to determine whether we can correlate changes in the surface morphology with changes in the density and binding capacity. Figure 9 shows representative topography images of films prepared with diglyme and triglyme, and statistical measurements derived from these images are shown in Figure 10. The diglyme(25) Birnie, D. P. J. Non-Cryst. Solids 1997, 218, 174-178.
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Figure 9. 50 × 50 µm topography images of the same films that are considered in Figure 8. All images are shown with the same 750-nm color scale.
Figure 10. (A) RMS roughness plotted as a function of spin rate. (B) Characteristic length scale vs spin rate.
based formulation is characterized by a monotonic decrease in both the roughness and λh with increasing spin rate. Surprisingly, both of these values actually increased with spin rate for the triglyme-based formulation. The above observations suggest that rough films with large λh tend to have the best binding properties. However, it is interesting to examine whether this trend persisted when the composition of the porogenic mixture was varied. Roughness, λh, and the autoradiography signal all exhibited nonmonotonic trends with increasing PVAc concentrations, and all of these curves shifted to lower concentrations when higher molecular weights were used. However, the line shapes are quite different for each set of data, indicating poor correlations between them. One might expect that the binding properties of the films would correlate more strongly with their specific surface areas. Using nitrogen adsorption porosimetry, we have shown that this holds true for MIPs that were prepared as bulk monoliths (unpublished results). In the current study,
however, the small amounts of material limited us to density measurements, from which we were able to derive estimatesof the fractional pore volume (which can be as high as 60% for the film prepared with 10% PVAc500).26 Preparing Thin Films. One of the parameters that we have not yet varied is the solvent/monomer ratio. This ratio was kept at 4/3 (v/v) throughout this study. However, we have found that by diluting the pre-polymerization mixture, it is possible to achieve much smaller film thicknesses. For example, a reaction mixture that contained 2.5% monomers diluted with 10% PVAc140 in diglyme produced an 18-nm film (as determined by ellipsometry) on a gold-coated glass substrate. Diluting the monomers with excess porogenic solvent changes the composition of the mixture, however, and one potential way to avoid this is by starting with a mixture similar in composition to what we have used thus far and diluting it with a secondary, highly volatile solvent such as toluene or chloroform. The rapid evaporation of the secondary solvent leaves behind the low-volatility components, so that the composition of the reaction mixture is, for practical purposes, unchanged. In addition, we suggest that such an approach to formulation would be particularly useful for processing techniques that require a mixture with a low viscosity to pump the liquid through a narrow capillary. Thus, we can envision using techniques such as ink-jet or dip-pen nanolithography to pattern surfaces with micron or submicron MIP features. Summary A novel technique was presented for preparing MIP films of controlled thickness and porosity, together with a thorough analysis of the morphology of these films. The method involved the in situ photolysis of functional and cross-linking monomers in the presence of a molecular template, while spinning the substrates at high speeds. The morphology of films produced with traditional imprinting mixtures were (26) While it is possible to estimate the surface area from AFM images, we believe that such measurements are misleading because of the inability of the tip to probe those pores that are beneath the surface but are still accessible for rebinding. The situation is further complicated by the convolution of the sample topography with the finite dimensions of the tip, so that holes that have aspect ratios larger than that of the tip cannot be accurately mapped. As a result, the binding curve data did not correlate very well with estimates of the surface area that were derived from AFM data.
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nonporous (and therefore exhibited small binding capacities) for two reasons: (1) the high evaporation rate of the solvent and (2) the rapid rate of polymerization compared to the kinetics of pore formation. Overcoming this kinetic barrier to pore formation was achieved by increasing the quench depth during polymerization via the addition of a linear polymer to the pre-polymerization mixture. The existence of a well-defined characteristic length scale in AFM images of the films revealed that the MIP separated from the porogen via spinodal decomposition. Increasing the concentration of the porogen or increasing its molecular weight led to a more advanced stage of phase separation. Autoradiography revealed optimum enhancement of the binding properties at moderate concentrations of PVAc. By varying the spin rate at a fixed composition, it is possible to produce films whose thickness varies over an order of magnitude. In the present case, we demonstrated this for films ranging in thickness from ca. 1 to 10 µm. However, films that are less than 100-nm thick can be readily prepared by diluting the pre-
Schmidt and Haupt
polymerization mixture with additional porogenic solvent. Although the reaction mixtures described herein are rather viscous, potential difficulties in processing can be overcome by diluting the mixture with a secondary, volatile solvent that evaporates shortly after deposition, thus opening the door to a wide variety of additional coating techniques. Acknowledgment. The authors acknowledge financial support from the European Union (MENDOS network) and from the Swedish Foundation for Strategic Research (BIOMICS network). We are also grateful to the Wenner-Gren Foundation for their generous financial support of R.H. Schmidt. Special thanks go to Goran Klenkar and Christopher Blanford for helping us with the profilometry measurements. Finally, we would like to thank Reine Wallenberg and the Department of Materials Chemistry at Lund University for allowing us to use their AFM. CM048392M
