Temperature-Sensitive Swelling of Poly(N-isopropylacrylamide

Article pubs.acs.org/Langmuir

Temperature-Sensitive Swelling of Poly(N-isopropylacrylamide) Brushes with Low Molecular Weight and Grafting Density Eva Bittrich, Sina Burkert, Martin Müller, Klaus-Jochen Eichhorn, Manfred Stamm, and Petra Uhlmann* Leibniz Institute of Polymer Research Dresden, Hohe Strasse 6, D-01069 Dresden, Germany S Supporting Information *

ABSTRACT: Temperature-sensitive poly(N-isopropylacrylamide) (PNIPAAm) brushes with different molecular weights Mn and grafting densities σ were prepared by the “grafting-to” method. Changes in their physicochemical properties according to temperature were investigated with the help of in situ spectroscopic ellipsometry and in situ attenuated total reflection Fouriertransform infrared (ATR-FTIR) spectroscopy. Brush criteria indicate a transition between a brush conformation below the lower critical solution temperature (LCST) and an intermediate to mushroom conformation above the LCST. By in situ ellipsometry distinct changes in the brush layer parameters (wet thickness, refractive index, buffer content) were observed. A broadening of the temperature region with maximum deswelling occurred with decreasing grafting density. The brush layer properties were independent of the grafting density below the LCST, but showed a virtually monotonic behavior above the LCST. The midtemperature ϑhalf of the deswelling process increased with increasing grafting density. Thus grafting density-dependent design parameters for such functional films were presented. For the first time, ATR-FTIR spectroscopy was used to monitor segment density and hydrogen bonding changes of these very thin PNIPAAm brushes as a function of temperature based on significant variations of the methyl stretching, Amide I, as well as Amide II bands with respect to intensity and wavenumber position. No dependence on Mn and σ in the wavenumber shift of these bands above the LCST was found. The temperature profile of these band intensities and thus segment density was found to be rather step-like, exceeding temperatures around the LCST, while the respective profile of their wavenumber positions suggested continuous structural and hydration processes. Remaining buffer amounts and residual intermolecular segment/water interaction in the collapsed brushes above the LCST could be confirmed by both in situ methods.

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INTRODUCTION For the development of smart surfaces, high attention is focused on stimuli-responsive polymers.12 Since type and rate of response to environmental stimuli can be regulated by chain length, composition, architecture, and topology, polymers offer a variety of opportunities to design such stimuli-responsive surfaces. One of the best studied environmentally responsive polymers, the water-soluble poly(N-isopropylacrylamide) (PNIPAAm), undergoes a temperature-sensitive phase transition in aqueous solution at its lower critical solution temperature (LCST) of ca. 31 °C.3−5 This transition is referred to as a coil-to-globule transition and takes place over a narrow temperature range of 1−2 K. In recent years, PNIPAAm thin films were investigated for various applications such as the control of protein adsorption and cell adhesion,6−8 solute separation9 or controlled drug delivery.10 Especially, well-defined brush films are very promising for the design of protein-resistant, temperaturesensitive interfaces, for example, used in sensor applications. In © 2012 American Chemical Society

the brush conformation, PNIPAAm with a low polydispersity proved to be virtually resistant toward protein adsorption.11,12 An increasing but still very small adsorbed amount of protein with decreasing grafting density could be found and was attributed to the possibility of proteins to penetrate the brush layer at low grafting densities.12 The theory of protein adsorption for these neutral water swellable brushes is a current topic of research,13,14 since there are also reports on the adsorption of considerable amounts of proteins to collapsed PNIPAAm brushes utilized in microfluidic devices15 or for protein release.16 PNIPAAm brushes with a low polydispersity of the grafted chains were prepared successfully by “grafting-to” of preformed chains to a reactive surface,11 and by “grafting-from” using Received: October 28, 2011 Revised: January 11, 2012 Published: January 12, 2012 3439

dx.doi.org/10.1021/la204230a | Langmuir 2012, 28, 3439−3448

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EXPERIMENTAL SECTION Materials. Carboxy-terminated poly(N-isopropylacrylamide) with different molecular weight (PNIPAAm-COOH) (see Table 1) and poly(glycidyl methacrylate) (PGMA, Mn =

surface-initiated atom transfer radical polymerization (ATRP).12,17 To purposefully design application-oriented temperaturesensitive interfaces, e.g., for long-living coatings in sensor/ microfluidic arrays, it is important to understand the influence of the brush structure parameters molecular weight Mn and grafting density σ on the temperature-sensitive swelling behavior. This swelling behavior was studied for PNIPAAm “graftingfrom” brushes with a variety of methods including neutron reflectometry,17 contact angle measurements,12,18,19 surface plasmon resonance,18 ellipsometry, 12 and atomic force microscopy (AFM).19 The temperature-sensitive deswelling of PNIPAAm brush films is generally characterized by an increase of the hydrophobicity of the brush surface above the LCST, the collapse of the polymer chains, and changes in the vertical density profile of the brushes. Moreover, a distinct transition was found for high molecular weights and intermediate to high grafting densities with all methods, but no collapse of the PNIPAAm chains by force−distance measurements and no sharp increase of hydrophobicity was found for low molecular weights and low grafting densities.19,20 With neutron reflectometry, a maximum of conformational changes was observed for intermediate grafting densities and high molecular weights.17 Also the monomer concentration profile was investigated with neutron reflectometry.17,21,22 Here a continually decreasing PNIPAAm volume fraction with height above the substrate could be found below the LCST, and the formation of two different density regimes with a dense region close to the substrate and a dilute region in the outer brush zone was found at the LCST. The formation of this two phases at the LCST also depended on Mn and σ. Above the LCST, the sharpness of the brush−solution interface increased again. These changes in the brush density profile with temperature could be connected successfully to self-consistent field simulations.23,24 For PNIPAAm “grafting-to“ brushes, to our knowledge there exist only few detailed studies on the temperature sensitivity.20,25 These brushes are prepared with well-defined preformed polymer chains that can be designed and functionalized according to the needs of application before grafting. Furthermore, very high switching amplitudes of homopolymer and binary “grafting-to” brushes in the intermediate grafting regime could be proven.11,26 Thus this type of brush is appealing for an easy, cheap, and fast design of functionalized surfaces with broad application possibilities. Herein we report on very thin PNIPAAm brushes (dry thicknesses from 3 to 24 nm) with relatively low molecular weight and grafting density prepared by the “grafting-to“ approach, and on their temperature-sensitive swelling behavior at different structure levels. At first, the dry PNIPAAm brushes were characterized by ellipsometry and AFM to prove the brush character as well as the homogeneous coverage of the surface with the brush polymer. Secondly, changes in the total swollen brush thickness and the volume percent of buffer inside the brush layers were monitored systematically for one molecular weight by in situ spectroscopic ellipsometry. Finally, on the molecular scale, changes in the hydrogen bonding of the grafted chains and water molecules were examined for the first time with in situ attenuated total reflection-Fourier-transform infrared (ATR-FTIR) in such thin brush films.

Table 1. Characteristics of Carboxy-Terminated PNIPAAm Used for the Brush Preparation sample PNIPAAm-COOH

(M̅ n)/(g/mol)

(M̅ w)/(M̅ n)

N

PN132k PN47k PN45k PN28k

132 000 47 600 45 000 28 500

1.29 1.22 1.72 1.46

1168 421 398 252

17,500 g/mol, Mw/Mn = 1.7) were purchased from Polymer Source, Inc. (Canada). All organic solvents were purchased from Merck KGaA (Germany), dried according to established procedures, and freshly distilled before their use. For ellipsometric measurements, polished single-crystal silicon wafers of {100} orientation with a native SiOx layer thickness of ca. 2 nm were used for substrates. To perform ATR-FTIR measurements, trapezoidal silicon internal reflection elements (Si-IRE, 50 × 20 × 2 mm3) from Komlas GmbH (Germany), allowing incident angles of θ = 45° were used. Prior to the grafting of polymers, all silicon substrates were treated with absolute ethanol for 15 min in an ultrasonic bath and were exposed to a cleaning solution (1:1:5) of deionized water (Millipore, Germany), ammonium hydroxide (28%), and hydrogen peroxide (35%) (Acros Organics, Germany) at 70 °C for 20 min. Afterward, they were rinsed in deionized water repeatedly and dried under nitrogen flux. “Grafting to” of Carboxy-Terminated PNIPAAm. Primarily a solution of 0.02 wt % PGMA in tetrahydrofuran (THF) was spin coated (Spin 150, SPS Coating, The Netherlands) onto the silicon substrates and heated for 20 min in a vacuum oven at 100 °C to react the silanol groups of the substrate with a fraction of the epoxy groups of PGMA, thus forming an anchoring layer equipped with the remaining epoxy groups for the following “grafting to” process.27 Afterward, a 1 wt % solution of PNIPAAm-COOH in THF was spin-coated onto the anchoring layer and annealed in a vacuum oven at 150 °C for several time intervals to obtain grafted brush layers with different grafting densites attached to the PGMA via ester bondings. Noncovalently bonded polymer was finally removed by Soxhlet extraction for 2.5 h in THF. Characterization Methods. ATR-FTIR Spectroscopy. ATR-FTIR spectroscopy was applied to characterize PNIPAAm brush layers in the dry state and in contact with 0.01 M phosphate buffered saline (PBS) solution (Aldrich, Germany) at a constant pH of 7.4. Therefore a commercial ATR-FTIR attachment operated with the single-beam-sample-reference (SBSR) concept (OPTISPEC, Zürich, Switzerland) was used.28−30 The ATR-FTIR attachment was installed on the IFS 55 Equinox FTIR spectrometer (BRUKER-Optics GmbH, Ettlingen, Germany) equipped with globar source and mercury cadmium telluride (MCT) detector. A home-built transparent in situ cell (M.Müller, IPF Dresden, Germany) was used, which sealed the upper sample (S) half and the lower reference (R) half of the Si-IRE by oval O-rings forming S- and Rcompartments, respectively, which both can be filled with various aqueous solutions. (Figure 1a,b). In this work, the brush layer was located solely on the S half of the Si-IRE, and the R 3440


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Figure 1. Experimental in situ ATR-FTIR (a,b) and in situ ellipsometry setup (c), both equipped with facilities to heat the buffer solutions.

was controlled. Heating and cooling cycles for each sample were performed between 15 and 45 °C with a heating/cooling rate of 0.2 °C/s, and up to three cycles were measured. The volume fraction of buffer inside the swollen PNIPAAm brush layer was modeled by an effective medium approach (EMA). In this approach, the effective dielectric function (refractive index n) of the heterogeneous layer is described on the basis of the known dispersion relations for n of the two components with varying individual volume fractions.35 The EMA according to Bruggeman was used, where the following condition has to be fullfilled:

half was uncoated. The SBSR concept implies that singlechannel spectra IS,R(ν) of the S and R half of the Si-IRE were recorded separately by guiding one IR beam alternately through these halfs. Normalizing the single-channel spectra according to A(ν) = −log(IS(ν)/IR(ν)) resulted in absorbance spectra (A(ν)) with proper compensation of the background absorptions due to the SiOx layer, solvent, water vapor (spectrometer), and ice on the MCT detector window. The temperature of the in situ cell was controlled by a homebuilt heating jacket, whose temperature could be adjusted via a thermostat water bath. Each measurement was done after an annealing time of 30 min. Ellipsometry. A spectroscopic ellipsometer (M2000, Woollam Co., Inc., Lincoln NE, USA) equipped with a rotating compensator was used to measure the ellipsometric data Δ (relative phase shift) and tan Ψ (relative amplitude ratio) of the brush films in the dry state as well as in situ in 0.01 M PBS solution at pH 7.4 within a batch cuvette (TSL Spectrosil, Hellma, Muellheim, Germany) (Figure 1c).31 Dry film measurements were performed at angles of incidence Φ0 of 65°, 70°, and 75° in the wavelength range of 371−1679 nm. For the in situ ellipsometry, Φ0 was fixed at 68° to achieve irradiation of the sample perpendicular to the cuvette sides. The wavelength range for in situ measurements was reduced to 371−800 nm. To evaluate the refractive index n and thickness h of the PNIPAAm brushes, a multilayer-box-model consisting of silicon, silicon dioxide, anchoring layer PGMA, and a PNIPAAm brush was assumed. The dispersion relations for silicon and silicon oxide were taken from the literature,31 and the refractive index of PGMA was set to 1.525.32 For the PNIPAAm brush layer, either a Cauchy relation (n(λ) = A + B/ λ2) was used to describe the dependence of the refractive index on the wavelength for this nonabsorbing polymer, or, in the case of thin films below 10 nm thickness, the refractive index was fixed to n = 1.47 because of a strong correlation between n and h in this region of film thickness.11 As an example, the Cauchy relation for a PN47k brush with a grafting density of 0.21 nm−2 is n(λ) = 1.453 + 0.006/λ2. Swollen brushes were also modeled with the multilayer box model, since more complex models could not be applied successfully for these thin brush films. However, complex models are used for much thicker polymer films in the literature.33,34 For the monitoring of the temperature-dependent swelling/deswelling of the PNIPAAm brushes, the temperature of the buffer solution was adjusted by a home-built heating stage equipped with testPoint software and the actual temperature at the brush surface

2 − n2 nbuffer

2 − n2 n polymer

+ fpolymer 2 0 = fbuffer 2 nbuffer + 2n2 n polymer + 2n2

(1)

Here f buffer and f polymer are the volume fractions of buffer and polymer in the mixed layer, respectively. nbuffer, npolymer, and n represent the dispersion relations of the refractive index for the buffer solution, the dry polymerm, and the mixed (swollen) layer. n(λ) for the buffer was measured by a temperaturecontrolled digital multiple wavelength refractometer DSR-k (Schmidt + Haensch GmbH and Co., Berlin, Germany). For the mixed layer as well as the dry polymer layer, Cauchy relations were used as described above. The Bruggeman approach is based on the assumption of a random mixture of polymer and buffer components with volume fractions of the same magnitude. No host medium can be assigned to one of the components. Atomic Force Microscopy. Root-mean-square roughnesses (rms) of the polymer brush films was determined by AFM (Nanoscope IIIa-Multimode Microscope, Veeco, USA). Tips of the type “BSTap” (Budget Sensors, Bulgaria) with a resonance frequency of 300 kHz and a spring constant of 40 N/m were used for tapping mode imaging.

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RESULTS AND DISCUSSION In the following, properties of thin PNIPAAm brush films in both the dry state and in contact with aqueous media are reported. At first, PNIPAAm brush films were characterized in the dry state by AFM and ellipsometry to determine brush parameters such as roughness, thickness, refractive index, grafting density, and surface concentration of the dry film, as well as to calculate brush criteria for good (below the LCST) and bad (above the LCST) solvent conditions. Then for these PNIPAAm films, the temperature-sensitive swelling behavior in 3441


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whether the prepared PNIPAAm films are brushes, there exist brush criteria in the literature.12,36 The distance between grafting sites is compared to twice the radius of gyration Rg of a free chain, leading in the case of direct comparison to the brush criterium of dg/(2Rg) ≪ 1,12 but also a comparison to the area πRg2, which a free chain would occupy, is possible and leads to a difference in the calculated reference value of a small factor.36 We used the first brush criterion, which was already applied for PNIPAAm brushes, and determined Rg for good and bad solvent conditions given by the polymer solution theory.12,38 For good solvent conditions (e.g., PNIPAAm in water below the LCST), the distance between grafting sites dg is compared to the Flory radius RF = LN3/5. L is the size of a monomer, and N is the degree of polymerization, where we took L ≈ 0.3 nm from the literature.12 For bad solvent conditions, the radius of a collapsed chain RC = LN1/3 has to be considered in the brush criterion. Since RC is smaller than RF, the bad solvent conditions are the tighter conditions if the PNIPAAm films are in the brush regime. Strictly, the formulas for Rg at different solvent conditions were developed for classical neutral brushes with no specific interaction with the solvent molecules, and deviations exist for PNIPAAm brushes from the experimentally determined Rg up to 40%, where Rg is decreased below and slightly increased above the LCST compared to the classical theory.39 With respect to these results, the use of the persistence length rather than the monomer size to calculate Rg should be discussed. Additionally, there can be found slightly different values for the monomer size L in the literature.40 With respect to the brush criterion, all PNIPAAm films presented in this paper are in the brush regime below the LCST at good solvent conditions. At bad solvent conditions they are no longer in the brush but are presumably in the mushroom regime. Additionally the transition between brush and mushroom regime is affected by the statistics of grafting and the polydispersity of the brush polymers, leading to a less sharp transition and a larger zone of intermediate grafting densities between these two regimes.36 As can be seen later from Figure 3, the deswelling is not complete, but the brush quality should be considerably reduced above the LCST. Thus the deswelling process presented in the in situ measurements can be considerably influenced by a transition from the brush regime to an intermediate and to the mushroom regime. 2. Characterization of the Brush Film in Contact to Aqueous Solutions. The swelling of PNIPAAm brushes in buffer solution as a function of the temperature was investigated using two complementary measurement methods. Detailed structural information shall be obtained on changes in the brush thickness and the buffer content (vol %) in the brush layer (ellipsometry) as well as on the formation of hydrogen

contact with aqueous solutions was analyzed by ellipsometry and ATR-FTIR measurements. Especially, the influence of the molecular weight and of the grafting density was focused upon. 1. Characterization of the Dry Brush Films. 1.1. Scanning Force Microscopy. In Figure 2, a typical AFM image

Figure 2. AFM height image of a PN45k brush taken directly after preparation and extraction in THF. The film is proven to be homogeneous, and no island formation occurred. Additionally, the cross-section profile is displayed as an inset for a path of 400 nm marked in black.

of a PNIPAAm brush film (molcular weight of 45 000 g/mol) is shown. Obviously, smooth and uniform films were obtained by the “grafting to” approach, which was confirmed by values of rms of less than 1 nm (see Table 2). 1.2. Ellipsometry. The typical structural brush parameters grafting density σ, distance between grafting sites dg, and the surface concentration Γp were determined based on the ellipsometric dry layer thickness h using eqs 2−4,36 where ρ is the density, and NA is Avogadro's number. The density was taken for bulk PNIPAAm from the literature.37

Γp = ρh dg =

(2)

Mn NAhρ

(3)

σ = dg −2

(4)

The calculated data is presented in Table 2 for the highest grafting density achieved for each molecular weight. To decide

Table 2. Dry Layer Parameters (Thickness h, Surface Concentration Γp, Grafting Distance dg, Grafting Density σ), the Radii of Nongrafted Polymer Coils RF at Good and RC at Bad Solvent Conditions As Well As the Corresponding Brush Criteria, and the Roughness (rms) of Silicon Oxide, the PGMA layer, and Typical PNIPAAm Brush Filmsa

a

layer

hdry/nm

SiOx PGMA PN132k PN47k PN45k PN28k

1.8 ± 0.1 1.5 ± 0.2 10.1 ± 0.2 15.7 ± 0.3 7.5 ± 0.2 4.5 ± 0.2

ΓP/(mg/m2)

10.8 16.8 8.0 4.8

dg/(nm)

4.5 2.2 3.0 3.2

σ/nm−2

0.05 0.21 0.11 0.10

RF/nm

20.7 11.3 10.9 8.3

dg/2RF

0.11 0.10 0.14 0.19

RC/nm

3.2 2.2 2.2 1.9

dg/2RC

rms/nm

0.7 0.5 0.7 0.8

0.2 0.3 0.4 0.4 0.4 0.6

A bulk density of PNIPAAm of 1.07 g/cm3 was used for the calculations.37 3442


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Figure 3. Swollen brush thicknesses (a) and refractive indices at λ = 631.5 nm (b) as a function of temperature for PN47k brushes with different grafting densities swollen in PBS buffer solution. The evaluated data displayed is for the first cooling cycle, respectively. The error range of the swollen brush thickness did not exceed 5% of the total value. For the refractive index, the maximum error was less than 1%. Thus error bars were not added to the figures for a clearer presentation.

process. Furthermore, a decrease in the total layer thickness below and above the LCST and a broadening of the temperature region with maximum deswelling can be observed from a grafting density of 0.33 nm−2 down to 0.11 nm−2 (Figure 3a). For grafting densities lower than 0.11 nm−2, changes in the swollen brush thickness are virtually identical between 25 and 32 °C, as well as above the LCST for the grafting densities of 0.11 nm−2 and 0.07 nm−2. We attribute these identical swelling curves to a more individual chain behavior, where the PNIPAAm films are presumably no longer in a brush or an intermediate conformation. The decrease of the density of polymer chains at the surface with grafting density is reflected in the monotonous decrease of the refractive index below 0.11 nm−2, as presented at a wavelength of light of 631.5 nm for swollen and collapsed PN47k brushes in Figure 3b. For the increase in thickness at 0.04 nm−2 grafting density above the LCST, we still have no explanation, but a failing of the modeling due to a very low amount of polymer at the surface is possible in this case. Refractive indices above the LCST are always lower than for the dry polymer brushes as presented in Table 3, indicating remaining buffer in the collapsed brush state. When the grafting density is increased, refractive indices also increase below and above the LCST, except for the 0.33 nm−2 brush. For the latter, n above the LCST is slightly lower than that for the 0.21 nm−2 brush, consistent with a higher swollen brush thickness at the grafting density of 0.33 nm−2 (Figure 3a). We propose that this is an effect of the increased interchain interaction at this higher grafting density, hindering the deswelling process, where the PN47k film is still in a more brush-like conformation above the LCST. Temperature-sensitive changes in the buffer content (vol%) inside the swollen PN47k brushes with different grafting densities are shown in Figure 4. These volume fractions are calculated by an EMA approach from the dispersion relations known for the dry polymer brush, the buffer solution, and the swollen polymer brushes. Below the LCST at the beginning temperature of the measurements at 15 °C, the volume fraction of the buffer was 90 ± 5%, similar for all investigated grafting densities, which indicates highly swollen polymer brushes. Above the LCST, a differentiation in the buffer volume fraction according to the grafting density is visible. Here a general decrease in buffer content can be observed with increasing grafting density. The PN47k brushes with the highest grafting

bonds (ATR-FTIR) as a function of the brush parameters, grafting density, and molecular weight. 2.1. In Situ Spectroscopic Ellipsometry. Changes in the layer parameters swollen brush thickness, in situ refractive index, and volume percent of buffer inside the brush were investigated for one molecular weight (Mn = 47 600 g/mol) as a function of the grafting density by spectroscopic ellipsometry. Five different grafting densities were achieved by variation of the grafting time during annealing of the polymer layers. The corresponding dry layer parameters of PN47k brushes are listed in Table 3. The refractive index for all dry brushes was fitted Table 3. Dry Layer Parameters and Brush Criteria for Good and Bad Solvent Conditionsa hdry/nm

σ/nm−2

dg/nm

dg/2RF

dg/2RC

3.3 ± 0.3 5.2 ± 0.1 7.9 ± 0.3 15.7 ± 0.3 24.1 ± 0.4

0.04 0.07 0.11 0.21 0.33

5.0 3.8 3.0 2.2 1.7

0.22 0.17 0.13 0.10 0.08

1.1 0.9 0.7 0.5 0.4

a

Grafting density and distance between grafting sites were calculated from the dry thickness according to eqs 3 and 4. The Flory radius of RF = 11.3 nm and the radius of a collapsed coil of RC = 2.2 nm for PN47k from Table 2 was used to calculate the brush criteria.

from the ellipsometric data and was n(631.5 nm) = 1.47 ± 0.01. Only for the brush with the lowest dry layer thickness of 3.3 nm was the refractive index fixed at 1.4 due to the correlation of n and h in the fit. To discuss changes in the temperature-sensitive deswelling with varying grafting density more closely, the swollen brush thicknesses derived from cooling experiments of several PN47k brushes are presented in Figure 3. High changes in the swollen brush thickness (Figure 3a) and the refractive index of the swollen layer (Figure 3b) can be achieved with these “grafting-to” brushes. For example, the thickness of a brush with a grafting density of 0.21 nm−2 can be reduced about 80% above the LCST. These remarkably high changes in the brush parameters are in our opinion a result of the decrease in the quality of brush conformation at elevated temperature as stated in Table 3. Thus the PNIPAAm chains act more like individual chains with increasing temperature, and interchain interactions are less important for the swelling 3443


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maxima indicate a small reswelling of the PNIPAAm films after an initial collapse, and this behavior was not observed so far. The response of the brush systems to changes in temperature lead to changes in the brush parameters swollen brush thickness, refractive index and buffer content up to 80%. Since these changes in brush parameters occur in a relatively low temperature range, the midtemperature ϑhalf provides a design parameter for the application of these brush films in functional surface coatings. 2.2. ATR-FTIR spectroscopy. Results of in situ ATR-FTIR measurements are shown in Figure 6. ATR-FTIR spectra of PNIPAAm brushes grafted on the Si IRE are given in the dry state, swollen at T = 24 °C and collapsed at T = 50 °C for the molecular weights 132 000, 45 000, and 28 000 g/mol. Generally, these spectra on thin PNIPAAm brush films agreed with IR spectra on PNIPAAm in solution,42 featuring the intense Amide I (mainly ν(CO)) between 1625 and 1650 cm−1 and Amide II (mainly δ(NH)) band between 1535 and 1555 cm−1 due to the amide groups as well as the CH-stretching region between 3000 and 2800 cm−1 mainly due to the isopropyl groups as prominent bands. The small peak at 1730 cm−1 corresponds to the ν(CO) band due to the ester group formed between telechelic COOH group of PNIPAAm and PGMA. The wavenumber positions of Amide I and II of PNIPAAm are highly indicative of the type and degree of hydrogen bonding as a function of the temperature (T) as it was described for PNIPAAm solutions therein.43−47 For T < LCST, where PNIPAAm is claimed to be in the coiled state, the wavenumber position of the Amide I appears lower and that of the Amide II band higher than for T > LCST, where PNIPAAm is claimed to be in the aggregated globule state. Lowering the Amide I band position can be assigned to hydrogen bond formation between the amide CO and water (C O···H···O−H) due to weakening CO double bond character, and enhancing the Amide II band position is due to H bond formation between amide N−H and water (N−H···O−H2), restricting the δ(NH) bending mode and increasing its force constant. Wavenumber positions of the PNIPAAm brush film as a function of sample state, temperature, and molecular weight are given in Table 4. Generally, significant shifts of the Amide bands could be obtained for the different sample states. An upward shift from 1627 (1628) to 1635 cm−1 and a downshift from 1558 to 1553 cm−1 was obtained for the Amide I and Amide II band, respectively, upon raising the temperature from T = 25 to 50 °C. Moreover, no significant influence of the molecular weight was obtained on that transition behavior, suggesting no lateral hindrance from grafting density aspects of the surface attached chains. With regard to the brush criteria presented in Table 2 this is an indication of a conformational change from PNIPAAm brushes below the LCST to intermediate or mushroom conformation with considerably less interchain interaction above the LCST. Interestingly, an upward shift to 1647 cm−1 and a downshift to 1535 cm−1 of Amide I and Amide II band, respectively, was obtained for the dry PNIPAAm brush compared to the wet state for both T = 25° and even 50 °C. This suggests that the PNIPAAm chains in the dry state are presumably in a poorly hydrated globule state. Furthermore, significant shifts of the position of the assymmetric CH3-stretching vibration, denoted as νas(CH3), due to the isopropyl groups could be obtained for the studied samples, which are also given in Table 4. All obtained band shifts were reversible.

Figure 4. Calculation of the percental content of PBS buffer in the swollen PN47k brushes as displayed in Figure 3 as a function of temperature and grafting density. The error range of the buffer content did not exceed 5% of the total value.

densities of 0.21 nm−2 and 0.33 nm−2 showed a remaining buffer content above the LCST of 24% and 32%, respectively. The latter higher buffer content for the 0.33 nm−2 brush is consistent with its higher swollen brush thickness (Figure 3a) and its lower refractive index (Figure 3b) above the LCST, supporting assumptions on the role of brush structure in the swelling process as discussed above. In light-scattering studies at latex particles grafted with PNIPAAm, similar water contents of 90% below the LCST and 25% above the LCST were found,41 which are in good agreement with our results. Finally the mid-temperature ϑhalf of the deswelling process was evaluated. This temperature was taken at the median refractive index between its minimum at 15 °C and its maximum above the LCST (Figure 5). Here also differences due to heating or cooling of the brush surfaces are presented.

Figure 5. Dependence of the midtemperature ϑhalf of the deswelling process evaluated from refractive index data in PBS buffer solution on the grafting density of PN47k brushes. ϑhalf from heating and cooling experiments are compared.

As can be seen, an increase in ϑhalf with increasing grafting density was observed. Differences upon heating and cooling the brush surface were highest for intermediate grafting densities, indicating a grafting density dependent hysteresis. The curves were fitted to parabola functions as a guide for the eye. ϑhalf is affected by the maxima in the refractive index observed for some grafting densities in Figure 3b. These 3444


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Figure 6. In-situ ATR-FTIR spectra on the ν(CH) band region (left image) and amide band region (right image) of PNIPAAm brush films in the dry state (a), swollen at 24 °C (b), and collapsed at 50 °C (c) in PBS buffer for three different molecular weights: 28.000 g/mol (solid line), 45.000 (dashed line), 132.000 g/mol (dotted line), respectively. Lines are drawn at the peak maxima of the spectra of collapsed films at 50 °C, respectively.

Table 4. Wavenumber Positions of νas(CH3) and Amide I and II Peaks of PNIPAAm Brushes at Different States wavenumber position [±1 cm−1] swollen state at 24 °C

dry state

collapsed state at 50 °C

peak

PN132k

PN45k

PN28k

PN132k

PN45k

PN28k

PN132k

PN45k

PN28k

νas(CH3) Amide I Amide II

2970 1647 1536

2970 1647 1535

2971 1647 1535

2981 1627 1558

2980 1627 1558

2980 1628 1558

2976 1635 1552

2976 1635 1553

2975 1635 1552

Figure 7. In situ ATR-FTIR spectra on the ν(CH) band region (a) and amide band region (b) of PNIPAAm (132.000 g/mol) brush films in contact to PBS buffer at the temperatures (from bottom to top) recorded at the temperatures 22, 26, 28, 30, 32, 34, 35, 38, and 44 °C. Lines are drawn at the peak maxima of the 22 °C spectrum, respectively.

water molecules are in the vicinity of the isopropyl group, yet postulating hydrogen bonding between water and C−H bond (C−H···OH2), which is supported by ab initio calculations.48 Hence we conclude for the moment that in the deyhydration mediated coil/globul transitions, the isopropyl groups are also involved. To have a more detailed view on this temperature-induced hydration/conformation process, the temperature-induced

Concerning that, the PNIPAAm brush in the wet state showed a downshift from 2981 (2980) cm−1 for T = 25 °C to 2975 (2976) cm−1 for T = 50 °C and further to 2970 cm−1 for the dry sample; again independently of the molecular weight. An analogous downshift of the νas(CH3) position for PNIPAAm solution upon heating above LCST was reported by Maeda.45 They explained this effect, i.e., an upward shift upon cooling below LCST, by contraction of the C−H bond, if 3445


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Figure 8. Integrals (a) and wavenumber positions (b) of the νas(CH3), Amide I, and Amide II bands in ATR-FTIR spectra (Figure 7) on thin PNIPAAm films as a function of temperature.

on curve fitting the νa(CH3), Amide I, and Amide II band regions by two components, respectively, is given in detail in the Supporting Information. Therein, the wavenumber shift upon temperature increase could be explained by the varying intensity of these two underlying components, which can be assigned either to internal hydrogen bonding within NIPAAm segments or external hydrogen bonding between segment and water, for each of the three bands. Most importantly, even for the low-temperature ATR-FTIR spectrum of the PNIPAAm brush film, two Amide I (Amide II) components were needed, while for the low-temperature FTIR spectra of PNIPAAm in solution, only one component was needed to obtain appropriate fits. Hence obviously, PNIPAAm in solution can be fully hydrated, unlike PNIPAAm as a brush film. On the other hand, two components were also found in the hightemperature ATR-FTIR spectrum, which suggests a persistent fraction of water molecules not to be released from their NIPAAm segments. Those are probably associated with a tight hydration shell. Furthermore, plotting the component integrals versus temperature, continuous courses were obtained. Light scattering studies at latex particles grafted with PNIPAAm showed that the water content of the swollen polymer is 90 wt % (50 water molecules per NIPAAm monomer).41 At the phase transition, the macromolecule collapses with a remaining water content of 25 wt % (2 water molecules per monomer). By ATR-FTIR as well as in situ spectroscopic ellipsometry (discussed above) we made similar observations. In the ATRFTIR results, the peak positions of the Amide I and II bands in the collapsed state were not consistent with the dry state (compare Figure 6 (left image) curves a and c). This is in accordance with our statement above, that some rest water always remained within the brush.

swelling of a PNIPAAm-132k brush brush (dry thickness: 15 nm) in contact with PBS buffer was studied at higher temperature resolution. The related ATR-FTIR spectra recorded between T = 24 and 44.5 °C with increments of ΔT = 2 °C around the LCST transition are shown in Figure 7a for the νas(CH3) band (isopropyl groups) and Figure 7b for the Amide I and II bands. From these spectra, both an overall increase in intensity and significant shifts of the wavenumber positions of νa(CH3), Amide I, and Amide II at around 2980, 1631, and 1556 cm−1, respectively, were found. In Figure 8a, the intensities (integrals) of these three bands are plotted versus temperature T. Constant integral values were obtained from T = 22 °C up to around T = 32 °C for both amide bands and up to around 35 °C for the νa(CH3) band, whereafter the band integrals were promptly increasing until T = 44 °C. Increases in the band integrals of spectra on PNIPAAm brush films measured by ATR-FTIR reflect a densification of polymer segments, which can be quantified by a modified Lambert−Beer law due to Fringeli.29 According to this reference and under the valid assumption of thin polymer films (herein 15 nm), band integrals scale linear to polymer segment concentration. So we conclude that there is an increase in NIPAAm segments with increasing temperature, which supports the increase of the refractive index with T for the PN47K found by ellipsometry shown in Figure 3b. To gain further insight into the thermoinduced structural changes in the PNIPAAm brush film, the wavenumber positions of the νas(CH3), Amide I, and Amide II bands were plotted versus the temperature in Figure 8b. Monotonous upshifts were obtained for the νas(CH3) and Amide I band positions, and downshifts were obtained for the Amide II band. Unlike the rather step-like temperature profile of the band integrals (Figure 8a), no such prompt increase was found for the wavenumber position profiles. From this finding, we qualitatively conclude that the structural change takes a more continuous course, whereas the segment concentration change takes a more jump-like one. To gain a more quantitative picture of the obvious structural changes, an alternative treatment of the ATR-FTIR data based

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CONCLUSIONS Temperature sensitivity at isotonic conditions was presented for PNIPAAm brushes with relatively low molecular weight and grafting densities prepared by the “grafting-to” method. These 3446


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suggesting a cooperative increase in NIPAAm segment concentration at higher temperatures, which is qualitatively in line with the ellipsometry results on the temperature profile of the refractive index. On the other hand, the courses of wavenumber positions and band components, which were assigned to certain hydrogen bonding types (internal segment/ segment or external segment/water), versus temperature suggested continuous structural and hydration processes, which is in line with theoretical predictions. At temperatures below the LCST, PNIPAAm brush films still contain intramolecular hydrogen bonds between segments, which is in contrast to the solution state. On the other hand, at temperatures above the LCST, PNIPAAm brushes still contain intermolecular hydrogen bonds between segments and water, which is confirmed by ellipsometry, identifying a remaining rest buffer content in the collapsed PNIPAAm films.

brushes are appealing for applications as long-living surface coatings, e.g., in microfluidic devices, as substrates for cell growth and harvesting, or in other fields where a precise actuation of interface properties such as layer thickness, density, or water content is necessary. It was confirmed that PNIPAAm “grafting-to” brushes have a similar temperature-sensitive swelling behavior as reported for “grafting-from” brushes in the literature. However, the phase transition theory for PNIPAAm brushes cannot be applied, because a transition from a brush conformation below the LCST to an intermediate to mushroom regime above the LCST seems likely and affects the swelling process. Novel information on the swelling process in PBS-buffer solution could be obtained by the in situ methods spectroscopic ellipsometry and ATR-FTIR. Spectroscopic ellipsometry was used to examine changes in the parameters thickness and refractive index of the swollen brush layers, and ATR-FTIR was used to observe changes in the vibration bands of amide and isopropyl goups. An increase of the temperature region with maximum deswelling was observed for decreasing grafting densities with in situ spectroscopic ellipsometry, pointing at a more heterogeneous transition at low grafting densities. Since deswelling of the PNIPAAm “grafting-to” brushes should be affected by changes in the quality of the brush state, we are of the opinion that with decreasing brush quality, i.e., at lower grafting densities and at elevated temperature, there is a less collective behavior of the PNIPAAm chains due to reduced interchain interactions. Taking a statistical distribution of grafting points at the surface into account, a more heterogeneous deswelling of PNIPAAm brushes seems likely with decreasing grafting density. The buffer content of the swollen brush layers was identical below the LCST, but depended on the grafting density above the LCST. For most of the brushes, the buffer content decreased with increasing σ, reflecting a higher density of deswollen chains at the surface. An exception was observed for the PN47k brush, the one with the highest grafting density. We assume that this points to the fact that the latter films have a brush conformation below and above the LCST, and the deswelling is reduced due to increased interchain interactions. Moreover, we regard the decrease in brush quality with increasing temperature as being responsible for the high “switching amplitude” observed in swollen brush thickness, refractive index, and buffer content, which is most desirable for applicational purposes. The mid-temperature of the deswelling process increased with increasing grafting density from 28.5 to 31.5 °C with the same general trend for heating and cooling the PNIPAAm brush films. Thus a grafting density-dependent design parameter for applicational purposes was found. A decrease of the degree of external hydrogen-bonding to water above the LCST was proven by in situ ATR-FTIR measurements in monitoring the temperature-sensitive shift of amide vibration bands, whereas the brushes were still considerably hydrated compared to the position of vibration bands in the dry state. No dependence of the studied IR band positions at 24 and 50 °C on molecular weight and grafting density could be observed, indicating a similar state of hydration of the individual polymer chains in the brush for all three investigated PNIPAAm brushes for each temperature respectively. The course of the ν(CH3), Amide I, and Amide II band integrals versus temperature was found to be rather steplike exceeding a certain temperature around 32−35 °C,

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ASSOCIATED CONTENT

S Supporting Information *

Curve fitting analysis of the Amide I, Amide II, and the ν(CH) band line shape by band components and plot of the intensities of the fitted band components versus temperature. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the Deutsche Forschungsgesellschaft (DFG) in the framework of the Sonderforschungsbereich 287 “Reaktive Polymere in Nichthomogenen Systemen, in Schmelzen und an Grenzflächen” within project B10 and in the DFG-NFS cooperation project “Design of new responsive materials based on functional polymer brushes for smart tuning and sensoring of proteins and particles adsorption” in the framework of the Materials World Network. We thank Roland Schulze for his help in the spectroscopic ellipsometry measurements.

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