Novel Thymine-Functionalized Polystyrenes for Applications in

Biomacromolecules 2004, 5, 1412-1421

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Novel Thymine-Functionalized Polystyrenes for Applications in Biotechnology. 2. Adsorption of Model Proteins Judit E. Puskas,* Yaser Dahman, and Argyrios Margaritis Macromolecular Engineering Research Center, Department of Chemical and Biochemical Engineering, The University of Western Ontario, London, Ontario, Canada N6A 5B9

Michael Cunningham Department of Chemical Engineering, Queen’s University, Kingston, Ontario, Canada K7L 3N6 Received November 26, 2003; Revised Manuscript Received March 11, 2004

This paper investigates the adsorption of bovine serum albumin (BSA) and bovine hemoglobin (BHb) model proteins onto novel thymine-functionalized polystyrene (PS-VBT) microspheres, in comparison with polystyrene (PS) microspheres. Maximum adsorption was obtained for both proteins near their corresponding isoelectric points (pI at pH ) 4.7 for BSA and 7.1 for BHb). FTIR and adsorption isotherm analysis demonstrated that, although both proteins were physisorbed onto PS through nonspecific hydrophobic interactions, adsorption onto the functionalized copolymers occurred by both physisorption and chemisorption via hydrogen bonding. FTIR analysis also indicated conformational changes in the secondary structure of BSA and BHb adsorbed onto PS, whereas little or no conformation change was seen in the case of adsorption onto PS-VBT. Atomic force microscopy (AFM), consistent with the isotherm results, also demonstrated monolayer adsorption for both proteins. AFM images of BSA adsorbed onto copolymers with 20 mol % surface VBT loading showed exclusively end-on orientation. Adsorption onto copolymers with lower functionality showed mixed end-on and side-on orientation modes of BSA, and only the side-on orientation was observed on PS. The AFM results agreed well with theoretically calculated and experimentally obtained adsorption capacities. AFM together with calculated and observed adsorption capacity data for BHb indicated that this protein might be highly compressed on the copolymer surface. Adsorption from a binary mixture of BSA and BHb onto PS-VBT showed good separation at pH)7.0; ∼ 90% of the adsorbed protein was BHb. The novel copolymers have potential applications in biotechnology. Introduction In the first part of a series of papers, we recently have reported the synthesis and characterization of novel polystyrenes (PS) carrying thymine functional groups via free radical emulsion copolymerization of styrene (St) and 1-(vinylbenzyl)thymine (1-VBT) in the presence and absence of divinylbenzene (DVB) in batch.1 Functionalizing polymers with nucleic acid bases is of particular interest because of their role in forming complexes between complementary bases. 1-VBT was introduced as a versatile monomer by Taylor et al.2,3 This monomer has a polymerization-active vinyl group, an amide group with pKa ) 9, two carbonyl groups available for hydrogen bonding, an aromatic ring for π-stacking, and a photoreactive double bond. Copolymerization of 1-VBT with St yielded thymine-functionalized PS, shown in Scheme 1. Narrowly distributed PS-VBT microsphere latexes with 1-4 mol % VBT content and 40-60 nm particle size were obtained. Freeze-drying of the latexes, which was used to preserve the integrity of the thymine moieties, followed by grinding yielded an average particle size of 2.07 µm with a particle size distribution of 1.6. Importantly, XPS analysis showed that the thymine content was much higher on the * To whom correspondence should be addressed.

Scheme 1. Structure of Novel PS-VBT Copolymers

surface of the particles (7-20 mol %) than in the bulk (1-4 mol %) measured by FTIR, NMR, and elemental analysis. We postulated that these novel microspheres could be very useful in biotechnology for applications such as affinity bioseparation and enzyme-linked immunosorbent assay (ELISA).4 The structure shown in Scheme 1 was expected to be advantageous for binding large molecules such as proteins, since the benzyl spacer group between the thymine and the polymer backbone gives more flexibility to the functional groups. This paper, the second in the series, reports the first results of our studies related to bioseparation.

10.1021/bm034497r CCC: $27.50 © 2004 American Chemical Society Published on Web 04/21/2004

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2. Adsorption of Model Proteins

Yoon et al.5-7 discussed the use of polymeric microspheres for the separation and purification of biomaterials. They pointed out that for effective separation the microspheres should have sizes in the micron range, with high porosity to obtain high load capacities. The use of sufficiently large polymeric microspheres produced by suspension polymerization failed to improve effectiveness, due to their inherently broad size distribution. Special techniques producing microspheres in the 30-400 micron range have been reported,8-13 but these involve extremely difficult, complicated, and expensive procedures. In addition, the additional attachment of affinity ligands specific for the given application is necessary. This latter approach led to effective separation even with submicron particles.10-14 It is known that protein adsorption results in the reduction of its conformational entropy, which must be compensated by sufficient favorable interaction between protein and surface.15 As shown in many studies, there are four forces responsible for protein adsorption onto solid surfaces:15,16 hydrophobic, van der Waals, ionic, and hydrogen bonding interactions, with pH and ionic strength playing major roles. Adsorption of serum proteins onto nonpolar microspheres such as PS17,18 and Teflon19 was shown to occur mostly by nonspecific interactions between the hydrophobic polymer surface and the protein. However, incorporating hydrophilic functional groups (PS/2-hydroxyethyl methacrylate and PS/acrylamide copolymers,17,18 PS/ metacrylic or acrylic acid copolymers,5 poly(vinyl chloride), and Nylon-6,619) gave rise to competing electrostatic and/or hydrogen-bonding forces.5,6,20 For instance, Shirahama and Suzawa17 found that increasing the acrylic acid or methacrylic acid comonomer content in PS microspheres resulted in increased BSA adsorption due to hydrogen bonding. Yoon et al.5-7 achieved effective separation of bovine serum albumin (BSA) from bovine hemoglobin (BHb) using submicron PS microspheres carrying carboxyl, amino, and sulfonate functional groups without ligand coupling, under specific conditions. They concluded that, when the hydrophobic PS surface is slightly modified with carboxylic or sulfonate groups, adsorption of BSA close to its isoelectric point (at pH ) 4.5) mainly occurs by hydrophobic interactions, whereas the adsorption of BHb (at pH ) 7) is inhibited by electrostatic repulsion. However, protein adsorption onto highly modified PS surfaces under the same conditions was governed by hydrogen bonding onto the carboxylic groups and by ionic interactions onto the sulfate groups of PS. These authors concluded that weakly modified surfaces were more efficient in terms of protein separation. The significance of this work is underlined by the fact that effective protein separation reportedly could only be obtained with proteins differing by at least a factor of 10 in molecular weight,21 whereas BSA and BHb have similar molecular weights. Very recently Causserand et al.22 reported the effective separation of these proteins by selective adsorption onto clay, exploiting electrostatic interactions. Against this background, it was of interest to investigate BSA and BHb adsorption onto our novel PS-VBT microspheres. This paper reports the results of adsorption isotherm, FTIR, and AFM studies, together with preliminary selective adsorption experiments.

Table 1. Characteristic Data of Polymer Microspheres Used for the Adsorption Studies Sw (m2/g)

VBT (mol %) sample i.d.

bulka

surfaceb

PS PS-VBT1-8 PS-VBT2-12 PS-VBT4-20

1.26 2.14 3.85

7.61 11.27 19.58

a 1H

F

(g/cm3)

calculated measured

1.04 1.26 1.24 1.23

2.84 2.26 2.30 2.38

2.91 2.43 2.52 2.59

NMR, FTIR, and elemental analysis1. b XPS1.

Table 2. Physical Characteristics of BSA and BHb22

molecular weight (Da) hydrodynamic dimensions (nm) equivalent radius (nm) isoelectric pH hydrophobic amino acid content (g/100 g protein) hydrophilic amino acid content (g/100 g protein)

BSA

BHb

67 000 14 × 4 × 4 3.61 4.7 44.07

68 000 7.0 × 5.5 × 5.5 3.10 7.1 54.16

56.16

43.13

Experimental Section Materials. Polystyrene (PS) and thymine-functionalized PS copolymers were synthesized, purified, and characterized as described previously.1 Table 1 lists the composition of copolymers used in this study. To facilitate sample preparation and AFM studies, only non-crosslinked soluble microspheres were used, which were shown to be equivalent to those slightly cross-linked with divinylbenzene (DVB).1 The acronyms in Table 1 represent copolymer bulk and surface composition. For instance, PS-VBT1-8 stands for 1 mol % VBT in the bulk and 8 mol % VBT on the surface. The polymers were freeze-dried to preserve the integrity of the heat-sensitive thymine and ground to yield an average particle size of 2.07 µm with a particle size distribution of 1.6. Table 1 also lists measured specific surface area and density data. Bovine serum albumin (BSA; fraction V) and bovine hemoglobin (BHb; both from Sigma Chemicals) were used as received. Table 2 summarizes physical properties of these proteins. Sodium acetate (98%, Caledon), acetic acid (99.7%, Caledon), Borax (sodium tetraborate) (99%, Sigma), hydrochloric acid (38%, BDH), sodium hydroxide (97%, Caledon), and toluene (99.8%, Aldrich) were used as received. Distilled and deionized (DDI) water was used for buffer solution preparation. 0.01 M phosphate buffer saline of pH 7.0 (PBS; Sigma Immuno Chemicals) was used as received. Procedures. Particle Size and Surface Area Measurements. The average particle diameter (Dn) and the size distribution of the polymer samples in Table 1 were measured by Malvern Mastersizer 2000 (Malvern Instrument, Ltd) and optical microscopy (Zeiss Axioplan, interfaced with a highresolution color video camera). Polymer densities were measured according to ASTM D 792-00. The specific surface area (Sw; m2/g) was measured using the BET method (Gemini 2360, Micromeritics Inc.; N2, T ) -196 °C, P ) 10-6 Torr), and was also calculated using eq 123 Sw )

6 FDn

(1)

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where Dn is the average particle diameter and F is the density (Table 1). The calculated Sw values agreed well with the BET data, as shown in Table 1. Protein Adsorption. Batch BSA and BHb adsorption experiments were conducted in 4 mL vials by adding 0.25 g of selected copolymer samples to 3 mL of the protein solutions prepared from corresponding stock solutions (3 g/L ) 44.4 µmol/L) prepared with sodium acetate (pH 3.5-5.5), PBS (pH 7.0), and Borax (pH 8.5-10.5) buffers. Specific concentration data will be given in the text and table and figure captions. All buffer concentrations were kept at 0.01 M, which is equivalent to solution ionic strength in the range of 0.01-0.02 M. Previous studies showed that in this range ionic strength has a negligible effect on adsorption.5,6,19,20 The vials were shaken for 24 h in an incubator shaker (New Brunswick Scientific Co. Inc.) at 25 ( 1 °C to ensure adsorption equilibrium. Then the solutions were allowed to settle for 1 h and centrifuged at 5000 rpm (Model CL, International Equipment Co.). The supernatant was filtered through low protein adsorption cellulose nitrate membrane filters (0.2 µm, Micro Filtration Systems, USA), and the polymer samples were dried in a vacuum desiccator overnight. BSA and BHb solution concentrations were measured using UV-visible spectroscopy (Varian Cary 50-Bio, Varian Inc.), based on calibration curves. BHb exhibits strong UVabsorbance at both 406 and 280 nm, whereas BSA shows a single maximum at 280 nm and negligible absorbance at 406 nm. BHb concentration was determined directly from the 406 nm adsorption, whereas BSA concentration was determined from the adsorption at 280 nm, after subtracting the contribution of BHb as determined from the concentration detected at 406 nm. The amount of protein adsorbed per unit weight of polymer (qe; g protein/g polymer) was calculated from eq 2 (C0 - Ce)V qe ) W

(2)

where V is the volume of buffer solution (L), C0 is the initial protein concentration, Ce is the protein concentration at adsorption equilibrium (g/L), and W is the weight of the adsorbent (g). Diffuse Reflectance Infrared Spectroscopy (DRIFTS) Studies. Fourier transform infrared spectroscopy in diffuse reflectance mode (FTIR-DRIFT) was performed using solid proteins, and dried polymer samples before and after equilibration in BSA and BHb solutions, all diluted to 10 wt % in KBr. FTIR-DRIFT spectra were recorded using a Bruker IFS 55 instrument equipped with a mercurycadmium-telluride (MCT) detector cooled with liquid nitrogen and a Spectra-Tech Baseline Drifts accessory. Spectra processing was performed using the Grams/32 software (Galactic Industries Co.). Atomic Force Microscopy (AFM) Studies. AFM images of thin polymer films before and after incubation in protein solutions were recorded in air at room temperature using a Nanoscope III multimode scanning probe microscope from Digital Instruments (Santa Barbara, CA). The polymer thin films were prepared by spin-coating 3 g/L polymer solutions

in toluene onto thin glass sheets using a headway resist spinner at 6000 rpm for 30 s. Three films of each sample were prepared. The polymer film thickness was ∼150 nm, measured by scratching the polymer film and measuring the vertical height in the AFM images between the surface of the glass sheets and the surface of the polymer. The glass sheets were shaken at 25 ( 1 °C in 10 mL solutions containing BSA or BHb (specific conditions will be given in the text and figure and table captions) and then dried in a vacuum desiccator overnight. To avoid any damage that may occur to biological specimens such as proteins,24 AFM was operated in the tapping mode using microfabricated cantilevers with a spring constant of 30 N/m. The probes consisted of a single-crystal silicon tip, with a nominal tip curvature of 5-10 nm, mounted on a single-beam cantilever with a resonance frequency of ∼300 kHz. To ensure that tip-sample interactions remain constant throughout the imaging procedure, the driving amplitude of oscillation was maintained constant at 60-70% of the free-air amplitude of 1.6 V that corresponds to approximately 50 nm of actual cantilever oscillation. Height and phase images were recorded at 1 Hz and 512 samples per line for 1 µm scan size. For the analysis of the observed surface structures, the Nanoscope image processing software was employed. The AFM height or phase images were collected from randomly selected sites and were found to be reproducible for the same polymer sample under the same experimental conditions. The vertical distances between lowest and highest-lying plateaus (hills and valleys) on the sample surface were measured for the determination of the thickness of adsorbed protein layer. Results and Discussions Adsorption Studies. As discussed above, protein adsorption onto PS and similar nonpolar polymer surfaces was shown to occur by hydrophobic interactions.17-19 Introduction of functional groups such as thymine, capable of specific interactions such as hydrogen bonding, is expected to result in additional adsorption. It should be mentioned here that microspheres prepared by emulsion polymerization, including the investigated PS-VBT microspheres, most likely contain surfactant residues and initiator fragments with hydroxyl groups, but the concentration of these residues is very small compared to the functional group concentration so they are usually not considered in adsorption studies. However, their roles in possible in vitro applications cannot be ignored. Figure 1 shows adsorption capacity plots for BSA and BHb on PS and PS-VBT4-20 at an initial protein concentration of C0 ) 0.25 g/L. It can be seen that the functionalized polymers show a higher adsorption capacity than PS in the entire pH range. The higher adsorption affinity of BHb to PS can be explained by its more hydrophobic nature.25 Protein adsorption onto both the PS and the functionalized copolymer PS-VBT420 reaches a maximum near the corresponding pI points, i.e., pH ) 4.5 for BSA and pH ) 7.0 for BHb. PS-VBT1-8 and PS-VBT2-12 had similar plots (not shown). Maximum protein adsorption at pI has been observed by previous



Figure 1. Effect of pH on the adsorption of BSA and BHb on PS and PS-VBT4-20. C0 ) 0.25 g/L, T ) 25 °C. Table 3. Adsorption Capacities of the Microspheres Listed in Table 1 (C0 ) 0.25 g/L, T ) 25 °C)

qe BSA

BHb

sample i.d.

mg/g polymer

mmol/mol VBTa

mg/g polymer

mmol/mol VBTa


1.25 2.26 2.69 3.35

0.022 0.022 0.021

1.78 2.75 3.07 3.82

0.021 0.020 0.020

a

Calculated by subtracting the amount of protein adsorbed onto PS.

researchers on various polymer surfaces such as PS13-24 and PS copolymers carrying different functionalities such as liposaccharide,26 methacrylic acid (MAA),4 and sodium styrene sulfonate (NaStS),5 in addition to multiblock copolymers with PS and poly(ethylene oxide) (PEO)27 sequences. Similar phenomenon was found on inorganic surfaces such as montmorillonite clay.22 Maximum protein adsorption near the corresponding pI was explained by the protein molecules being in neutral form,28 which reduces potential electrostatic

repulsion forces between them and they can assume a more condensed conformation. The lower adsorption capacities below and above the corresponding pI can be attributed to electrostatic repulsion between the charged protein molecules and/or the surface.13-22 Decreasing protein adsorption onto PS-VBT4-20 at pH > 9.5 can be related to additional repulsion forces between the negatively charged thymine moieties (pKa ) 9) and negatively charged BSA or BHb molecules.29 Table 3 lists adsorption capacities (qe) obtained for all copolymer samples listed in Table 1 at C0 ) 0.25 g/L. After subtracting the corresponding amounts adsorbed onto PS, the functionalized polymers adsorbed the same amount of protein on a molar basis (0.02 mmol protein/mol), indicating specific chemisorption. Figure 2 shows representative plots for the equilibrium adsorption of BSA and BHb onto PS at their corresponding pI, fitted to the linearized form of the Freundlich isotherm model represented by eq 330 log qe ) log qm + 1/n log Ce

(3)

where qm is the maximum capacity (mg protein/g polymer) and n is an exponential parameter representing the affinity of adsorption.30 Similarly good fits were obtained at pH ) 3.5, 4.5, 5.5, 7.0, 8.5, 9.5, and 10.5, while the Langmuir model did not describe the isotherms at any of the pH tested. The good fit to the Freundlich model indicates that both BSA and BHb adsorption onto the PS surface is governed by nonspecific hydrophobic interactions. Maximum adsorption capacity values (qm) obtained from the intercept of the plots are listed in Table 4. The qm of 6.37 and 8.06 mg protein/g polymer (equivalent to 2.19 and 2.77 mg protein/m2 polymer) for BSA and BHb, respectively, agree well with literature data; Revilla et al.31 reported 2.35 mg BSA/m2 PS, whereas Shirahama et al.32 reported 2.80 mg BHb/m2 PS. The higher adsorption capacity of BHb over BSA represents its higher affinity toward the nonpolar PS surface, consistent with results shown in Figure 1.

Figure 2. Linearized Freundlich isotherm model for BSA and BHb adsorption onto PS. C0 ) 0.125-3.0 g/L, pH ) 4.5 for BSA and 7.0 for BHb, T ) 25 °C.

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( )

Table 4. Maximum Adsorption Capacities (qm) of the Microspheres Listed in Table 1

qm BSA sample i.d.

mg/g mg/m2

PS 6.37 2.19 PS-VBT1-8 18.15 7.47 PS-VBT2-12 22.73 9.02 PS-VBT4-20 28.16 10.87

1 1 1 1 ) + qe Kqm C 1/n qm e

a

BHb mmol/mol mmol/mol mg/g mg/m2 VBTb VBTb 0.26 0.26 0.22

8.06 2.77 19.23 7.91 23.92 9.49 30.03 11.59

0.25 0.25 0.22

a Calculated from the Freundlich model (eq 3) for PS and from the Langmuir-Freundlich model for PS-VBTs (eq 4). b Calculated after subtracting the amount of protein adsorbed onto PS.

Figure 3. Linearized Langmuir-Freundlich isotherm model for BSA and BHb adsorption onto PS-VBT4-20. C0 ) 0.125-3.0 g/L, pH ) 4.5 for BSA and 7.0 for BHb, T ) 25 °C.

The data for BSA and BHb adsorbed onto functionalized PS-VBT copolymers did not show good fit with either the Langmuir or the Freundlich isotherm models but could very well be described using the linearized form of the combined Langmuir-Freundlich model represented by eq 430

(4)

where K is the adsorption constant. Figure 3 shows representative plots. The good fit shown in Figure 3 indicates that BSA and BHb adsorb to functionalized PS-VBT copolymers via a combination of nonspecific physisorption to PS sites and specific chemisorption onto thymine sites through hydrogen bonding. Similarly good fit was obtained at pH) 3.5, 4.5, 5.5, 7.0, and 8.5. Table 4 summarizes qm values calculated from eq 4 for BSA and BHb adsorption at their corresponding pI. Similarly to Table 3, qm values calculated after subtracting the corresponding amounts adsorbed onto PS were practically identical on a molar basis for all PS-VBT copolymer samples studied, i.e., ∼0.22-0.26 mmol protein/mol VBT. This proves similar adsorption stoichiometry for BSA and BHb onto thymine moieties at their corresponding pI. qm for PS-VBT4-20 was 10.87 mg/m2 (28.16 mg/g) for BSA and 11.59 mg/m2 (30.03 mg/g) for BHb. Yoon et al.5-7 reported 5.8 mg/m2 (0.03 mmol BSA/mol carboxyl) for PSPMMA microspheres and 12 mg/m2 (0.19 mmol BSA/mol) sulfate on PS-NaStS. These authors concluded that protein adsorption was governed by hydrogen bonding onto the carboxylic groups and by ionic interaction onto the sulfate groups of PS. We investigated the nature of the chemisorption onto the thymine functionalities of our copolymers using FTIR. FTIR Studies of BSA and BHb Adsorption. Figure 4a shows the FTIR spectra of solid BSA in KBr. In this spectrum, three major IR bands appear at 1645, 1535, and 3300 cm-1. The 1645 cm-1 (amide-I) band corresponds to the CdO stretch weakly coupled with C-N stretch and N-H bending, the 1535 cm-1 (amide-II) band represents C-N stretch strongly coupled with N-H bending, and the 3300 cm-1 band is assigned to N-H stretching

Figure 4. FTIR studies of BSA adsorption. (a) Solid BSA; (b) BSA on PS; (c) BSA on PS-VBT4-20 (polymer spectra subtracted). 10% solids in KBr; adsorption conditions: C0 ) 1 g/L, pH ) 4.5, T ) 25 °C.



Figure 5. FTIR studies of BHb adsorption. (a) Solid BHb; (b) BHb on PS; (c) BHb on PS-VBT4-20 (polymer spectra subtracted). 10% solids in KBr; adsorption conditions: C0 ) 1 g/L, pH ) 4.5, T ) 25 °C.

Figure 6. AFM images of polymers spin-coated onto Si (3% toluene solution, 6000 rpm) (a) PS; (b) PS-VBT4-20.

vibrations.32-34 The weak peak in the region of 1200-1300 cm-1 is assigned to N-H in plane bending coupled with C-N stretching and also includes C-H and N-H deformation vibrations.32-34 Figure 4b shows the IR spectrum of BSA adsorbed onto PS, after subtracting the PS spectrum. The amide-I peak shifted to 1677 cm-1, whereas no change was observed in amide-II and N-H peaks. Noinville et al.34 studied BSA adsorption onto nonpolar talc and polar montmorillonite. He assigned a 1681 cm-1 band to the “free” carbonyls in extended internal structures, which are in hydrophobic, random, or bent domains, whereas another band at 1670 cm-1 was assigned to the “free” carbonyls in more polar internal domains. Considering these assignments, the shifted peak at 1677 cm-1 in Figure 4b indicates that interactions between BSA and PS are mainly hydrophobic. It was suggested that BSA adsorbs by rearranging its hydrophobic domains toward the PS surface; thus, the orientation of the protein molecule changes.34 Figure 4c represents BSA on PS-VBT4-20, after subtracting the copolymer spectrum. In this figure, the amide-I band shifted

to 1660 cm-1. Again, no change was observed in the amideII and N-H bands. Noinville et al.34 assigned a 1660 cm-1 band to hydrogen bonded CdO in bundled internal R-helices, and a peak at 1651 cm-1 to hydrogen bonded CdO in external R-helices. Thus, our FTIR results indicate that BSA chemisorbed onto the thymine moieties through hydrogen bonding. The capability of PS-VBT to hydrogen bond with acidic protons was also demonstrated earlier by FTIR and proton NMR studies with phenol as a model compound.1 In the BHb spectrum (Figure 5a), the amide-I, amide-II, and N-H stretching vibration bands appear at 1645, 1544, and 3300 cm-1.32-34 In Figure 5, parts b and c, the amide-I peak shifted to 1677 and 1661 cm-1, respectively, whereas no shifts were observed in the amide-II band or N-H stretching. Similarly to BSA adsorption, we interpreted the FTIR results showing that BHb is physisorbed onto PS through hydrophobic interactions, whereas it chemisorbed onto the thymine moieties of the PS-VBT copolymers through hydrogen bonding. In addition, the FTIR data indicate conformational

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Figure 7. AFM images of BSA on PS; (a) phase image; (b) 3D image. Adsorption conditions: C0 ) 1.0 g/L, pH ) 4.5, T ) 25 °C.

Figure 8. Schematic diagram for possible orientations of adsorbed BSA.

changes of the BHb upon adsorption onto the nonpolar PS surface. Further investigations will be necessary to assess the consequences of conformational changes. AFM Studies. The adsorption of BSA and BHB was visualized using AFM. The surface morphologies of PS and PS-VBT4-20 are shown in parts a and b of Figure 6, respectively. PS has a smooth surface with almost no features, whereas PS-VBT4-20 shows some surface roughness with a vertical height of ∼1 nm. Figure 7, parts a and b, shows the surface morphology and the three-dimensional image of BSA adsorbed on PS. Figure 7 clearly shows BSA adsorbed on the surface with uniform vertical height of ∼5 ( 1 nm. Comparing this vertical height with the hydrodynamic dimensions of BSA (Table 2) indicates that BSA is oriented in a side-on mode. Figure 8 shows the two possible orientations for BSA, side-on and end-on, as reported in several studies.5,6,18,24,25,37,38 In the images of BSA adsorbed on PS-VBT1-8, two different surface patterns can be distinguished, with vertical heights of ∼5 ( 1 and 15 ( 1 nm (Figure 9). These most likely represent adsorbed BSA molecules in mixed end-on and side-on orientation. Similar results were obtained on PS-VBT2-12. Figure 10 shows BSA adsorbed on PS-VBT4-20, featuring only average vertical heights of ∼15 ( 1 nm. This can be assigned to an exclusively endon orientation. Interestingly, images obtained for BSA adsorbed onto PSVBT4-20 at pH values higher or lower than pI showed two

different surface patterns, indicating a mixed orientation such as that shown in Figure 9. The thickness of the adsorbed BSA layers (δ, nm) was also calculated using the following empirical equation5,6,37,38 δ)

3x3 qm π Fprotein

(5)

where 3x3/π is the “packing factor”. Equation 5 was first reported by Chiu and co-workers37,38 and was cited again by Shirahama and Suzawa32 as an approximate method to calculate the mean nominal thickness of adsorbed BSA protein layers, assuming the conservation of molecular volume and incorporating geometrical analysis of BSA.37-38 Table 5 lists thickness data calculated using eq 5. The calculated 5 nm thickness of the BSA layer on PS indicates a side-on orientation, in agreement with the AFM data. The layer thicknesses of 9 and 11 nm calculated for BSA on PS-VBT1-8 and PS-VBT2-12 indicate mixed end-on and side-on orientations. The δ value of 15 nm for PS-VBT4-20, on the other hand, signals exclusively endon orientation. On the basis of similar thickness calculation, Yoon et al.5,6,7 reported that BSA adsorbed onto slightly sulfonated PS in the side-on orientation, whereas the endon orientation was found in the case of highly sulfonated PS. The mechanism of adsorption was a combination of nonspecific hydrophobic and electrostatic interactions. Ex-



Figure 9. AFM images of BSA on PS-VBT1-8; (a) phase image; (b) 3D image. Adsorption conditions: C0 ) 1.0 g/L, pH ) 4.5, T ) 25 °C.

Figure 10. AFM images of BHb on PS-VBT4-20; (a) phase image; (b) 3D image. Adsorption conditions: C0 ) 1.0 g/L, pH ) 4.5, T ) 25 °C. Table 5. Comparison of the Measured and Calculated Thickness of Adsorbed BSA Layers (δ) at pH ) 4.5 δ (nm) BSA

a

sample i.d

calculateda

measuredb


5 9 11 15

5 5/15 5/15 15

Equation 5. b AFM images (standard deviation of ( 1 nm).

clusively, the end-on orientation was found on carboxylated PS, with a mechanism of a combination of hydrophobic and hydrogen-bonding interactions. The AFM images of BHb adsorbed on PS showed ∼6 ( 1 nm vertical height. Considering that BHb is nearly spherical with an approximately 6 nm diameter (Table 2), this indicates monolayer coverage. Interestingly, vertical heights of ∼3 ( 1 nm were observed in the case of BHb on PS-VBT samples. It has been reported that BHb is highly compressible.37 The equivalent radius of BHb (the minimum possible radius of an impenetrable spherical shape of the globular

protein) was reported to be 3.1 nm.36 Our AFM images thus indicate that BHb adsorbed onto PS-VBT may be highly compressed. Adsorption capacities of BSA and BHb onto polymer samples investigated in this study can be calculated assuming monolayer coverage of the surface. The maximum theoretical close-packed monolayer capacities (Ntheo) of BSA and BHb were calculated based on their hydrodynamic dimensions and was 8.9 × 1016 (end-on) and 2.1 × 1016 molecule/m2 (sideon) for BSA and 3.7 × 1016 molecule/m2 for BHb (BHb is close to spherical (Table 2) so only a single orientation was considered). The experimental monolayer adsorption capacities (Nexp) were calculated from qm in Table 4 and the specific surface area of the corresponding polymer samples listed in Table 1 and are summarized in Table 6. The data in Table 6 show, in agreement with the AFM images, that BSA most likely adsorbed onto PS in side-on orientation (Nexp ) 1.9 × 1016 molecule/m2). The values of Nexp for PS-VBT1-8 and PS-VBT2-12 indicate mixed side-on and end-on conformation. The Nexp ) 8.9 × 1016 molecule/m2 for PS-VBT4-20, on the other hand, indicates exclusively end-on orientation.

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Table 6. Measured Adsorption Densities of BSA and BHb at pH ) 4.5 and 7.0, Respectively

Nexpa(molecule/m2) (×10-16)

a

sample i.d.

BSA

BHb


1.9 6.7 8.1 8.9

2.4 7.0 8.4 10.0

Calculated from qm values in Table 3.

Figure 12. Adsorption from binary BSA-BHb mixtures onto PSVBT4-20. (a) mg/g; (b) fraction (0-1) of total adsorbed amount. Total protein concentration Cp ) 0.25 g/L, pH ) 7, T ) 25 °C.

Figure 11. Adsorption from binary BSA-BHb mixtures onto PSVBT4-20. (a) mg/g; (b) fraction (0-1) of total adsorbed amount. Total protein concentration Cp ) 0.25 g/L, pH ) 4.5, T ) 25 °C.

The Nexp ) 2.4 × 1016 molecule/m2 BHb on PS indicates less than monolayer coverage. On the other hand, the Nexp ) 7-10 × 1016 molecule/m2 for BHb on the PS-VBT copolymers would suggest possible multilayer coverage. This would be in direct contradiction with the adsorption models used. However, Ntheo based on the equivalent diameter of 3.1 nm is 13.2 × 1016 molecule/m2. Nexp is more consistent with this calculation. Similarly to our data, Kondo et al.39 reported 4.7 × 1016 molecule/m2 BHb adsorbed onto ultrafine silica gel particles, which is higher than the theoretically possible close-packed capacity of 3.7 × 1016 molecule/m2. They explained this with the high compressibility of BHb. Our data also indicate compression of the adsorbed BHb. Adsorption Studies in Binary mixtures. Adsorption experiments were conducted from binary mixtures of BSA

and BHb. Parts a and b of Figure 11 show respectively the amount (mg/g) and fraction (0-1) of BSA and BHb adsorbed onto PS-VBT4-20 at a total protein concentration of C0 ) 0.25 g/L at pH ) 4.5. These plots represent a close to ideal behavior with no preferential adsorption of either of the components. The total amount of proteins adsorbed remained nearly constant until about 0.75 weight fraction BSA, when it started to increase and reached the same value as in the single component adsorption studies. In contrast, at pH ) 7 very little BSA was adsorbed until it reached a weight fraction of 0.5 (Figure 12, parts a and b). In addition, the total amount of proteins adsorbed sharply dropped with increasing BSA content, compared to the single adsorption studies; at BSA ) 0.5, the total amount of adsorbed protein was 2.4 mg/g at pH ) 4.5, compared to 1.2 mg/g at pH ) 7. The depressed BSA adsorption at pH ) 7.0 can be explained by the electrostatic repulsion forces of negatively charged BSA molecules at pH ) 7.0, whereas BHb molecules exist in a neutral form. In addition, the BSA also seems to interfere with the adsorption of BHb, reducing its density on the surface. In single component BHb adsorption experiments, protein loading was higher than theoretical, which we explained with the high compressibility of this protein. In the presence of BSA at pH ) 7, this does not seem to happen. However, good separation with respectable loading was reached. Causserand et al.,22 who studied adsorption of BSA and BHb onto montmorillonite, reported


less than 1 mg/g total protein adsorption from a binary mixture at C0 ) 0.25 g/L for both pH 4.8 and 7, with a sharp decrease at pH ) 8.4. This study obtained good separation at pH ) 7.0. Thus, our PS-VBT copolymers have a potential to offer in bioseparation; more extensive studies are in progress in our laboratory. Conclusions In summary, we demonstrated that BSA and BHb were chemisorbed via hydrogen bonding onto the thymine functional groups of novel PS-VBT copolymers. FTIR spectra showed that conformational changes in the secondary structure of BSA and BHb occurred upon adsorption onto PS, whereas little or no conformation changes were observed upon adsorption onto PS-VBT copolymers. Adsorption capacities obtained experimentally and calculated theoretically together with AFM images proved that a monolayer of BSA adsorbed onto the copolymer with the highest VBT content (20 mol % on the surface) exclusively in end-on orientation. Adsorption capacities obtained experimentally for BHb onto PS-VBT copolymers were generally higher than the calculated capacities. This was explained by the compression of adsorbed BHb and was further confirmed by AFM images. Adsorption from binary mixtures of BSA and BHb onto PS-VBT showed good separation at pH ) 7.0, as up to 50 wt % BSA content BHb formed 90% of total adsorbed proteins. More extensive studies are in progress in our laboratories. Acknowledgment. The authors thank Dr. Yaohong Chen for her contribution to this paper. The Bayer-NSERC (Natural Sciences and Engineering Research Council of Canada) Industrial Research Chair, NSERC, and Bayer Polymers (Bayer Inc., Canada) are acknowledged for financial support. References and Notes (1) Dahman, Y.; Puskas, J. E.; Margaritis, A.; Merali, Z.; Cunningham, M. Macromolecules 2003, 36, 2198-2205. (2) Taylor, L. D.; Cheng, C. M.; Egbe, M. I.; Grasshoff, J. M.; Guarrera, D. J.; Pai, R. P.; Warner, J. C. J. Polym. Sci., Part A: Polym. Chem. 1995, 33, 2515-2519. (3) Grasshoff, J. M.; Taylor, L. D.; Warner, J. C. (Polaroid Corp.) Vinyl benzyl thymine monomers and their use in photoresists. U.S. Patent 5,455,349, Oct 3, 1995. (4) Bickerstaff, G. F. Immobilization of enzymes and cells; Human Press: Totowa, NJ, 1997. (5) Yoon, J.-Y.; Park, H.-Y.; Kim, J.-H.; Kim, W.-S. J. Colloid Interface Sci. 1996, 177, 613-620. (6) Yoon, J.-Y.; Kim, J.-H.; Kim, W.-S. Colloids Surf. A: Physicochem. Eng. Aspects 1999, 153, 413-419. (7) Yoon, J. Y.; Kim, J. H.; Kim, W. S. Colloids Surf. B 1998, 10, 356377. (8) Ugelstad, J.; Moerk, P. C.; Kaggerud, K. H.; Ellingsen, T.; Berge, A. AdV. Colloid Interface Sci. 1980, 13, 101-140.

Biomacromolecules, Vol. 5, No. 4, 2004 1421 (9) Vanderhoff., J. W.; El-Aasser, M. S.; Micale, F. J.; Sudol, E. D.; Tseng, C. J.; Siowanowicz, A.; Shen, H. R. ACS Polym. Mater. Sci. 1986, 54, 587-592. (10) Ugelstad, J.; Berge, A.; Ellingsen, T.; Schmid, R.; Nilsen, T. N.; Mork, P. C.; Stenstad, P.; Hornes, E.; Olsvik, O. Prog. Polym. Sci. 1992, 17, 87-161. (11) Colvin, M.; Chung, S. K.; Hyson, M. T.; Chang, M. J. Polym. Sci. A: Polym. Chem. 1990, 28, 2085-2095. (12) Hosoya, K.; Kishii, Y.; Kimata, K.; Araki, T.; Tanaka, N.; Svec., F.; Frechet, J. M. J. J. Chromatogr. A 1995, 690, 21-28. (13) Hosoya, K. and Ferchet, J. M. J. Polym. Sci. A: Polym. Chem. 1993, 31, 2129-2138. (14) Scopes, R. K. Protein Purification, 2nd ed.; Springer-Verlag: New York, 1990. (15) Lee, S. H.; Ruckenstein, E. J. Colloid Interface Sci. 1988, 125, 365379. (16) Haggerty, L.; Watson, B. A.; Barteau, M. A.; Lenhoff, A. M. J. Vac. Technol. B. 1991, 9 (2), 1219-1222. (17) Shirahama, H.; Susuki, K.; Suzawa, Y. J. Colloid Interface Sci. 1989, 129, 483-490. (18) Kondo, A.; Higashitani, K. J. Colloid Interface Sci. 1992, 150, 344351. (19) Absolom, D. R.; Zingg, W.; Neumann, A. W. J. Biomed. Mater. Res. 1987, 21, 161-171. (20) Kondo, A.; Higashitani, K. J. Colloid Interface Sci. 1992, 150, 344351. (21) Saksena, S.; Zydney, A. L. Biotech. Bioeng. 1994, 43, 960-968. (22) Causserand, C.; Kara, Y.; Aimar, P. J. Membr. Sci. 2001, 186, 165181. (23) Allen, T. Particle size measurement; Chapman and Hall: New York, 1990. (24) Oenich, M. D.; Warshawsky, A. Colloid Polym. Sci. 1991, 269, 139145. (25) Shirahama, H.; Suzawa, T. Colloid Polym. Sci. 1985, 263, 141146. (26) Delair, T.; Marguet, V.; Pichot, C.; Mandrand, B. Colloid Polym. Sci. 1994, 272 (8), 962-970. (27) Grainger, D. W.; Okano, T.; Kim, S. J. Colloid Interface Sci. 1989, 132, 161-175. (28) McMurry, J.; Organic Chemistry, 5th ed.; Brooks Cole: Belmont, CA, 1999. (29) Kurihara, K.; Abe, T.; Nakashima, N. Langmuir 1996, 12, 40534056. (30) Tien, C. Adsorption Calculations and Modeling; ButterworthHeinemann: New York, 1994. (31) Revilla, J.; El-Aissari, A.; Carriere, P.; Pichot, C. J. Colloid Interface Sci. 1996, 180, 405-412. (32) Shirahama, H.; Suzuki, K.; Suzawa, T. J. Colloid Interface Sci. 1989, 129 (2), 483-490. (33) Belfer, S.; Fainchtain, R.; Purinson, Y.; Kedem, O. J. Membr. Sci. 2000, 172, 113-124. (34) Noinville, S.; Revault, M.; Quiquampoix, H.; Baron, M. H. J. Colloid Interface Sci. 2000, 221, 273-283. (35) Iordanski, A.; Polischuk, A.; Zaikov, G. Macromol. Chem. Phys. 1983, 23, 33-59. (36) Singh, B. R. Infrared Analysis of Peptides and Proteins: Principles and Applications; ACS Symposium Series 750; American Chemical Society: Washington, DC, 2000. (37) Chiu, T. H.; Nyilas, E.; Leederman, D. M. Trans. Am. Soc. Artif. Int. Organs 1976, 22, 498-513. (38) Nyilas, E.; Chiu, T. H.; Herzlinger, G. A. Trans. Am. Soc. Artif. Int. Organs 1974, 20B, 480-90. (39) Kondo, A.; Fukuda, H. J. Colloid Interface Sci 1998, 198, 34-41.

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Novel Thymine-Functionalized Polystyrenes for Applications in

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