Thermoresponsive anionic block copolymer brushes with a strongly anionic bottom segment for effective interactions with biomolecules

Abstract

Brushes were prepared from a thermoresponsive strongly anionic block copolymer, namely poly(2-acrylamido-2-methylpropanesulfonic acid) (AMPS)-b-poly(N-isopropylacrylamide) (PAMPS-b-PIPAAm), by multistep atom-transfer radical polymerization with pH control of the reaction solution. The prepared block copolymer brushes were characterized using CHN elemental analysis, X-ray photoelectron spectroscopy, contact angle measurements, and zeta potential measurements. The results showed that dense polymer brushes were formed on the silica surfaces, and showed thermally modulated changes in their hydrophobic and anionic properties. Chromatographic analyses using silica beads modified with PAMPS-b-PIPAAm brushes as column-packing materials showed that the block copolymer brushes interacted more strongly with basic biomolecules than did brushes of a random copolymer, namely P(IPAAm-co-AMPS). PAMPS-b-PIPAAm copolymer brushes could therefore provide effective thermoresponsive anionic interfaces with strong anionic properties that could be modulated by changing the external temperature.

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Introduction

Stimuli-responsive polymers and their modified surfaces have attracted much attention because of their specific intelligent properties and wide range of new applications.^1–8 In particular, poly(N-isopropylacrylamide) (PIPAAm) and surfaces modified with this polymer are widely used in biomedical applications^1,2,9 because PIPAAm shows thermoresponsive hydrophilic/hydrophobic changes near body temperature (approximately 32 °C).¹⁰ Several biosensors have been developed based on the thermoresponsive properties of PIPAAm.^11–13 Thermoresponsive cell culture dishes for preparing cell sheets, i.e., monolayer cellular tissues,¹⁴ have been widely used in tissue engineering and regenerative medicine.^15–17 Thermoresponsive surfaces are also used in temperature-modulated bioseparation. One of their practical applications is in thermoresponsive chromatography,^18,19 in which PIPAAm-modified stationary phases are used as column-packing materials. The surface properties of the stationary phase can be changed by changing the external temperature because this changes the PIPAAm hydrophobicity, enabling modulation of the hydrophobic interactions between the stationary phase and analytes. The chromatographic system therefore does not require any organic solvent in the mobile phase, unlike conventional reverse-phase chromatographic systems. This prevents deactivation of bioactive compounds and reduces environmental pollution by organic solvents. The chromatographic system can be improved by improving the performance of the PIPAAm-modified stationary phase.¹⁸ Various methods for surface modification with PIPAAm have been investigated because the performance depends on the properties of the PIPAAm on the stationary phase, e.g., the length, density, and crosslinking.^18,20,21 These factors can be precisely controlled by using surface-initiated atom-transfer radical polymerization (ATRP) to modify stationary phases with PIPAAm.^22,23 ATRP can precisely control the molecular weight of a polymer, giving low polydispersity, and form polymer brush structures on substrates.^24–31 Surface-initiated ATRP has been used to investigate the use of PIPAAm and copolymer brushes in stationary phases for thermoresponsive chromatography.³² Thermoresponsive ionic copolymer brushes have been investigated for use in thermoresponsive ion-exchange chromatography. Various types of thermoresponsive ionic copolymer brushes for a range of thermoresponsive ion-exchange chromatographic applications have been prepared by incorporating appropriate ionic monomers into PIPAAm brushes.^33,34 The electrostatic and hydrophobic interactions between a thermoresponsive ionic copolymer and analytes can be modulated by changing the temperature.^33–36 Almost all types of thermoresponsive ionic copolymer brushes on chromatographic stationary phases have been prepared by incorporating ionic monomers into thermoresponsive polymer brushes by random copolymerization, resulting in a uniform distribution of ionic groups in the main PIPAAm chains. However, there is a limit to the amount of ionic monomer that can be incorporated into a thermoresponsive copolymer because an excessive amount of ionic monomer in the PIPAAm copolymer chains prevents copolymer phase transitions.^37,38 Electrostatic interactions between a copolymer and analytes is therefore limited in this type of thermoresponsive ion-exchange chromatography.

Block copolymerization by multistep ATRP would be an effective approach for grafting different types of functional block chains onto substrates because previous studies have shown that block copolymerization through ATRP can form functional block copolymer brushes by effective location of functional groups in the polymer brushes.^39–43 Block copolymerization enables the ionic and thermoresponsive chains to be separated in copolymer brushes. A large amount of ionic groups can therefore be introduced into copolymer brushes, leading to strong electrostatic interactions between analytes and the copolymer brushes. It has previously been reported that block copolymer brushes of poly(3-acrylamidopropyltrimethylammonium chloride) (APTAC)-b-PIPAAm are more strongly cationic than random copolymer brushes of P(IPAAm-co-APTAC).⁴⁴ Stationary phases modified with PAPTAC-b-PIPAAm brushes would be good candidates for thermoresponsive anion-exchange matrices for separation of acidic biomolecules. Therefore, thermally-modulated strongly anionic surfaces could be developed by preparing block copolymer brushes with strongly anionic base blocks and thermoresponsive upper blocks by block copolymerization. However, polymerization of anionic monomers is difficult because they deactivate the ATRP catalyst.^45,46 In block copolymerization, in particular, surface anionic groups of polymer brushes would prevent the second ATRP step.

In the present study, we used multistep surface-initiated ATRP to prepare block copolymer brushes consisting of thermoresponsive block chains and anionic block chains, namely poly(2-acrylamido-2-methylpropanesulfonic acid (AMPS)-b-PIPAAm), for thermoresponsive ion-exchange chromatographic separation of basic biomolecules through strong electrostatic interactions. Deactivation of the ATRP catalyst was prevented by adjusting the pH of the AMPS monomer solution to neutral in the first ATRP, and adding buffer solution to the IPAAm monomer solution in the second ATRP. The physicochemical properties of the prepared thermoresponsive anionic block copolymer brushes were investigated. The use of beads modified with block copolymer brushes in the elution of biomolecules was investigated.

Experimental

Materials

IPAAm was kindly provided by KJ Chemicals (Tokyo, Japan), and purified by recrystallization from n-hexane. AMPS was purchased from Tokyo Chemical Industry (Tokyo, Japan) and used as received. Acetone, methanol, 2-propanol [high-performance liquid chromatography (HPLC) grade], toluene (anhydrous), dichloromethane, formaldehyde, formic acid, tris(2-aminoethyl)amine (TREN), CuCl, CuCl₂, phosphate buffer (PB) powder (66.7 mM, pH 7.0), ethylenediamine-N,N,N′,N′-tetraacetic acid disodium salt dehydrate (EDTA·2Na), hydrophobic steroids, and catecholamine derivatives were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Basic proteins were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tris(2-N,N-dimethylaminoethyl)amine (Me₆TREN) was prepared using a previously described method.⁴⁷ Dialysis membrane tubing (molecular cut-off: 1000 kDa) was purchased from Spectrum Laboratories (Rancho Dominguez, CA, USA). [(Chloromethyl)phenylethyl]trimethoxysilane (CPTMS) was purchased from Gelest (Morrisville, PA, USA). Silica beads (specific surface area: 100 m² g⁻¹; diameter: 5 μm; pore size: 300 Å) were obtained from Chemco Scientific (Osaka, Japan). Glass beads (diameter 177–250 μm) were obtained from AS ONE (Osaka, Japan) and sieved using a 212 μm mesh. A stainless-steel column (50 × 4.6 mm inner diameter) was purchased from GL Science (Tokyo, Japan).

Copolymer brush grafting on silica beads

Silica bead surfaces were modified with an ATRP-initiator (CPTMS) using a previously reported method.⁴⁸ Briefly, silica beads were washed with hydrochloric acid at 90 °C and then beads (31.8 g) were placed in humidified air (relative humidity 60%) for 4 h. CPTMS (8.25 mL) was added to toluene (612.8 mL) and the solution was reacted with the silica beads for 16 h at 25 °C (Fig. 1).

Fig. 1 Scheme for preparation of PAMPS-b-PIPAAm-brush-grafted silica beads through surface-initiated ATRP.

Anionic thermoresponsive block copolymer brushes were prepared on the silica bead surfaces using multistep ATRP (Fig. 1). A typical polymerization procedure was as follows. AMPS (0.887 g, 4.28 mmol) was dissolved in water (21.4 mL) and the solution pH was adjusted to approximately 7.0 by adding 10 N NaOH solution. AMPS solutions of two concentrations (100 and 200 mM) were prepared by changing the amount of AMPS. AMPS solution (15.0 mL) and 2-propanol (134.8 mL) were mixed and the mixture was deoxygenated by argon gas bubbling for 1 h. CuCl (0.296 g, 3.00 mmol) and CuCl₂ (0.040 g, 0.299 mmol) were added under argon and the mixture was stirred for 10 min. Me₆TREN (0.774 g, 3.36 mmol) was added to the solution. The solution was reacted with ATRP-initiator-modified silica beads (3.5 g) at 25 °C for 1 h. After polymerization, the PAMPS-modified beads were rinsed with methanol and toluene, and dried in a vacuum oven at 50 °C for 2 h.

The second ATRP, for PIPAAm grafting on PAMPS brushes, was performed as follows. A mixture of 2-propanol and 66.7 mM PB was used as the reaction solvent to prevent deactivation of the ATRP catalyst by the acidic conditions associated with PAMPS brushes. 2-Propanol (38.5 mL) and 66.7 mM PB (pH 7.0, 4.28 mL) were mixed in a 100 mL flask, and IPAAm (1.46 g, 12.9 mmol) was dissolved in the solution. In this polymerization, various IPAAm concentrations were used, namely 300, 600, and 1000 mM. The IPAAm solution was deoxygenated by argon gas bubbling for 1 h. CuCl (0.0847 g, 0.848 mmol), CuCl₂ (0.0115 g, 0.0855 mmol), and Me₆TREN (0.221 g, 0.961 mmol) were added to the IPAAm solution under argon, and stirred for 10 min to form the ATRP catalyst. The PAMPS-brush-modified silica beads (1.0 g) were placed in a 50 mL glass vessel. The glass flask containing the ATRP reaction solution and the glass vessel containing the PAMPS-modified beads were placed in a glove bag. Oxygen in the glove bag was removed by evacuating three times, until the oxygen concentration in the bag was below 0.3%. The reaction solution was then poured into the glass vessel containing the PAMPS-modified beads, and the vessel was sealed. ATRP was performed by continuous shaking of the glass vessel at 25 °C for 16 h (Fig. 1). After the reaction, the beads were washed with acetone, methanol, 50 mM EDTA solution, and water, using an ultrasonic cleaner, and dried in a vacuum oven at 50 °C for 3 h.

Random copolymer brushes were prepared on silica beads to enable comparison of the properties of the block copolymer and random copolymer brushes. IPAAm (2.92 g, 25.8 mM) was dissolved in 2-propanol (38.5 mL) and the prepared 200 mM AMPS solution (4.28 mL) was added to the solution. The solution was deoxygenated by argon gas bubbling for 1 h. The ATRP catalyst, CuCl (0.0847 g, 0.848 mmol), CuCl₂ (0.0115 g, 0.0855 mmol), and Me₆TREN (0.221 g, 0.961 mmol) were added to the reaction solution. The solution was reacted with the ATRP-initiator-modified beads (1.0 g) at 25 °C for 16 h.

Beads modified with PIPAAm homopolymer brushes were prepared using a similar method. Briefly, IPAAm (2.92 g, 25.8 mM) was dissolved in 2-propanol (38.5 mL) and 66.7 mM PB (pH 7.0, 4.28 mL); the same amounts of CuCl/CuCl₂/Me₆TREN as for the preparation of random copolymer brushes were added to the solution. The solution was reacted with ATRP-initiator-modified beads at 25 °C for 16 h.

The same copolymer brushes were used to modify glass beads (average diameter: 212–250 μm) to investigate changes in the thermoresponsive surface wettability. The silica beads modified with copolymer brushes could not be used for contact angle measurements because the beads were small and unstable.

The same copolymer brushes were prepared on glass bead surfaces using a similar polymerization method, except that initiator-modified glass beads (2.0 g) were used instead of initiator-modified silica beads. The ATRP-initiator-modified glass beads were prepared using a previously reported method.⁴⁹

The samples were named based on the initial monomer and molecular concentrations in each ATRP step; for example, beads prepared using 10 mM AMPS solution were denoted by 10AS, and the beads prepared by subsequent ATRP using 600 mM IPAAm solution were denoted by 10AS-600IP. Copolymer brushes prepared by random copolymerization of 600 mM IPAAm solution and 20 mM AMPS solution were denoted by 600IP-r-20AS.

Copolymer brush characterization

The amount of initiator and grafted copolymer on the silica bead surfaces were determined by CHN elemental analysis of the prepared silica beads using an CHN elemental analyzer (PE2400, PerkinElmer, Waltham, MA, USA). The amount of initiator (g m⁻²) was calculated using the following equation:where % C_I is the carbon percentage obtained in the analysis, % C_I(calcd) is the calculated carbon percentage in the initiator molecules (CPTMS), and S is the surface area of the beads (100 m² g⁻¹).

The amount of copolymer on the silica beads (g m⁻²) was calculated as follows:

where % C_P is the increase in the carbon percentage from the initiator beads after ATRP, and % C_P(calcd) is the calculated weight percentage of carbon in the copolymer.

The grafted copolymer on the silica beads was removed using NaOH solution (10 mol L⁻¹) to dissolve the silica surface, and the molecular weight and polydispersity index of the obtained copolymer were determined using a gel-permeation chromatography (GPC) system (GPC-8020 II, Tosoh, Tokyo, Japan) equipped with serially connected columns (TSKgel SuperAW2500, TSKgel SuperAW3000, and TSKgel SuperAW4000, Tosoh). N,N-Dimethylformamide (DMF) containing 50 mM LiCl was used as the mobile phase. Calibration was performed using poly(ethylene glycol) standards.

The copolymer density on the bead surfaces was calculated as follows:

where m_C is the amount of grafted copolymer (g m⁻²), N_A is Avogadro's number, and M_n is the number-average molecular weight of the grafted copolymer.

The surface elemental composition of the silica beads was investigated using X-ray photoelectron spectroscopy (XPS; K-Alpha, Thermo Fisher Scientific, Waltham, MA, USA). Wide scans were performed to identify all elements present on the silica bead surfaces. Deconvolution of the C 1s peak was performed to obtain information about the chemical bonds.

The zeta potentials of the beads were measured using a zeta potential analyzer (Zetasizer Nano-ZS, Malvern, Malvern, UK). The copolymer-modified beads were suspended in 5 mM KCl solution at a concentration of 0.5 mg mL⁻¹. The zeta potential was measured at various temperatures.

The contact angles of the copolymer-brush-modified glass beads were measured using a previously reported method.^50,51 PB (66.7 mM, pH 7.0) was dropped onto a glass slide. A small amount of the copolymer-modified glass beads was placed on the PB droplet. A cover glass was placed on the droplet and beads, and the beads were squeezed to the liquid–air interface. The angle between the liquid–air interface and copolymer-modified beads in the liquid phase was measured to give the contact angle. The contact angle was measured at various temperatures.

Chromatographic analysis using basic biomolecules and proteins

The prepared silica beads were packed into an empty HPLC column (column length: 50 mm, inner diameter 4.6 mm), as previously reported.²² The bead-packed column was connected to an HPLC system (JASCO, Tokyo, Japan). PB (pH 7.0, 66.7 mM) was used as the mobile phase. The column temperature was modulated by immersing the column in a water bath, and the bath temperature was controlled using a water circulator (CTA 400, Yamato, Tokyo, Japan). The elution behaviors of catecholamine derivatives were examined to investigate the acidic properties of the copolymer brushes on the beads. L-3,4-Dihydroxyphenylalanine (DOPA) (0.43 mg), adrenaline (1.69 mg), and tyramine (1.95 mg) were dissolved in 2.7 mg mL⁻¹ Na₂SO₃ solution (6 mL). The elution behaviors of the catecholamine derivatives were monitored at 254 nm. Hydrophobic steroids were also used as analytes to investigate the hydrophobic properties of the copolymer brushes by observing the elution profiles. Samples were prepared by dissolving hydrocortisone (3.92 mg) and dexamethasone (5.03 mg) in ethanol (10 mL). Basic protein elution profiles were also observed because the elution profiles of proteins differ depending on their molecular weights and the basic properties provide information on the copolymer brushes. Samples were prepared by dissolving proteins (3.0 mg) in PB (pH 7.0, 66.7 mM, 6 mL), and the elution profiles were observed at 280 nm.

Results and discussion

Characterization of copolymer brushes on beads

The surface elemental compositions of the copolymer-modified beads were investigated using XPS; the results are summarized in Table 1, and Fig. 2 shows the C 1s peak deconvolution for each ATRP step in bead preparation; Fig. S1† shows the C 1s XP spectra of the prepared beads. A comparison of the results for the initiator-modified beads and PAMPS-modified beads shows that the latter had higher nitrogen and sulfur contents, indicating modification of the bead surfaces with PAMPS brushes. The carbon and nitrogen contents were increased by the second ATRP step. These results indicate that PIPAAm was grafted on PAMPS brushes. Deconvolution of the C 1s peaks also suggested that polymerization was successful in each step, because additional peaks attributed to the C=O bonds of PAMPS and PIPAAm were observed (Fig. 2 and S1†).⁵² Also, the silicon content of the beads modified with block copolymer brushes decreased with increasing IPAAm monomer concentration in the second ATRP step. This suggests that the length of the PIPAAm segment in the block copolymer brushes increased with increasing initial monomer concentration in ATRP, leading to an increase in the block copolymer brush layer thickness; XPS detection of the silica base layer was hindered by the increased block copolymer brush thickness. The sulfur contents quoted in Table 1 are not related to the amount of PAMPS in the copolymer structure. This is because the sulfur content of the copolymer is small, leading to measurement errors in such a small range.

Table 1 Elemental analyses of PAMPS-b-PIPAAm-brush-grafted silica beads using XPS with 90° take-off angle

Code^a	Atom^c (%)						N/C ratio
	C	N	O	Si	Cl	S
a All samples were named using the monomer abbreviation and feed molar concentration. “IP” and “AS” represent IPAAm and AMPS, respectively.b Estimated atomic composition of each monomer.c Data from three separate experiments are shown as mean ± SD.
Initiator modified	25.4 ± 7.42	0.17 ± 0.29	45.7 ± 2.06	26.1 ± 5.09	2.45 ± 0.26	0.14 ± 0.18	—
10AS	24.8 ± 2.60	2.25 ± 0.55	46.5 ± 3.29	24.7 ± 4.61	0.79 ± 0.33	0.96 ± 0.65	0.0910
20AS	24.1 ± 0.79	1.95 ± 0.36	45.2 ± 0.50	26.6 ± 0.41	1.03 ± 0.11	1.16 ± 0.64	0.0810
10AS-300IP	36.3 ± 3.73	3.94 ± 0.31	37.7 ± 0.38	20.0 ± 4.96	0.76 ± 0.41	1.32 ± 0.55	0.108
10AS-600IP	45.6 ± 0.51	6.80 ± 0.73	29.6 ± 0.72	16.1 ± 0.79	1.05 ± 0.40	0.91 ± 0.57	0.149
10AS-1000IP	60.6 ± 0.96	8.96 ± 0.77	20.5 ± 0.31	8.70 ± 0.53	0.62 ± 0.21	0.65 ± 0.57	0.148
20AS-300IP	32.4 ± 1.30	3.56 ± 0.62	40.0 ± 1.71	21.7 ± 2.32	0.95 ± 0.33	1.27 ± 0.40	0.110
20AS-600IP	45.2 ± 2.26	5.85 ± 0.61	30.9 ± 1.49	15.3 ± 1.03	1.27 ± 0.45	1.57 ± 0.72	0.129
20AS-1000IP	62.6 ± 0.52	9.64 ± 0.30	18.8 ± 1.54	7.32 ± 0.48	1.01 ± 0.56	0.70 ± 0.72	0.154
600IP-r-20AS	61.3 ± 1.11	9.17 ± 2.27	22.3 ± 4.80	5.60 ± 2.39	1.31 ± 1.12	0.34 ± 0.35	0.150
600IP	66.6 ± 3.59	9.85 ± 1.02	18.2 ± 4.30	4.32 ± 0.95	0.94 ± 0.46	0.11 ± 0.10	0.148
Calcd of IPAAm^b	75.0	15.0	15.0	—	—	—	0.167
Calcd of AMPS^b	53.9	8.33	33.3	—	—	5.56	0.155

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Fig. 2 Deconvolution of C 1s XPS peaks of prepared beads: (A) ATRP-initiator-modified silica beads, (B) 20AS, and (C) 20AS-1000IP (abbreviations are defined in Table 1).

Previous reports indicated that acidic monomers cannot be polymerized using ATRP catalysts because such monomers react rapidly with metal catalysts. Also, the pH of the reaction solution is important in maintaining the catalytic activity.^45,46 In the present study, we modulated the pH of the monomer solution in the first ATRP step, and phosphate buffer was added to the monomer solution to maintain the pH in the second ATRP step. These modulations enable polymerization of the AMPS and IPAAm monomers on PAMPS brushes.

Table 2 summarizes the GPC and CHN elemental analysis data for the block copolymer brushes. The carbon content of the copolymer increased in the first step of ATRP of AMPS, indicating that PAMPS was grafted onto the silica bead surfaces. The carbon content of the 20AS beads was larger than that of the 10AS beads; this is because the grafted PAMPS brush length increased with increasing AMPS concentration. The estimated amount of PAMPS on the bead surfaces was small. Our previous study indicated that strong sulfonic acid groups functioned as effective ion-exchange groups, although the amount in the copolymer brushes was small.³⁸ The small amount of PAMPS brushes was therefore sufficient to provide ion-exchange groups in the copolymer. The carbon content of the beads modified with PAMPS-b-PIPAAm brushes was higher than that of the PAMPS-brush-modified beads. This result indicates that PIPAAm was grafted onto the PAMPS brushes, leading to formation of a PAMPS-b-PIPAAm brush layer on the silica bead surfaces. The carbon content increased with increasing IPAAm monomer concentration in the second ATRP step, indicating that the length of the PIPAAm segment in the grafted block copolymer increased with increasing initial monomer concentration. These results are consistent with the XPS results.

Table 2 Characterization of PAMPS-b-PIPAAm-brush-modified beads

Code^a	Elemental composition (%)			Immobilized initiator^c (μmol m^−2)	Grafted polymer^c (mg m^−2)	Mn^d	Mw/Mn^d	Graft density (chains per nm^2)
	C^b	H^b	N^b
a All samples were named using the monomer abbreviation and feed molar composition of AMPS or IPAAm. “IP”, “AS” represent IPAAm and AMPS, respectively.b Determined by CHN elemental analysis (n = 3).c Estimated from carbon composition.d Determined by GPC using DMF containing 50 mmol L^−1 LiCl as a mobile phase; calibration curves were obtained using a PEG standard.
Initiator modified	5.10 ± 0.02	0.15 ± 0.13	0.83 ± 0.03	4.97
10AS	6.02 ± 0.01	0.07 ± 0.02	1.22 ± 0.05		0.255
20AS	6.30 ± 0.03	0.10 ± 0.04	1.32 ± 0.05		0.335
10AS-300IP	9.21 ± 0.07	0.21 ± 0.12	1.83 ± 0.06		0.851	7300	4.49	0.0703
10AS-600IP	14.0 ± 0.02	1.07 ± 0.09	2.80 ± 0.09		1.91	9300	3.08	0.124
10AS-1000IP	17.7 ± 0.05	1.74 ± 0.05	3.58 ± 0.13		2.86	13 300	3.32	0.129
20AS-300IP	8.76 ± 0.07	0.27 ± 0.05	1.89 ± 0.12		0.792	4600	3.64	0.105
20AS-600IP	13.6 ± 0.07	0.89 ± 0.05	2.80 ± 0.09		1.82	10 400	2.50	0.105
20AS-1000IP	18.4 ± 0.06	1.86 ± 0.12	3.75 ± 0.07		3.07	15 900	3.04	0.116
600IP-r-20AS	18.0 ± 0.03	1.70 ± 0.04	3.86 ± 0.03		2.90	12 500	1.87	0.140
600IP	19.2 ± 0.01	1.87 ± 0.05	4.05 ± 0.12		3.18	8600	2.00	0.222

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The copolymer was removed from the silica surfaces using NaOH solution, to enable direct measurement of the M_n values and polydispersity indexes (M_w/M_n, where M_w is the weight-average molecular weight) of the polymer brushes grafted on beads. The M_n of the retrieved PAMPS-b-PIPAAm increased with increasing IPAAm monomer concentration in the second ATRP step. The polydispersity index was large, although polymerization was performed using ATRP. This is attributed to the porous structure of the silica beads. Our previous study indicated that the polymer grafting conditions at the outer and inner pores of silica beads differ as a result of different feed rates of monomers and ATRP catalysts at polymerization sites.⁵³ Furthermore, the length of the grafted polymer on the inner pore was restricted by the inner pore diameter. These factors result in a large polydispersity index. The estimated graft density of the copolymer brushes was greater than 0.1 chains per nm², indicating that densely packed block copolymer brushes were formed on the silica bead surfaces.

The surface wettabilities of the copolymer brushes were investigated by measuring the contact angles of copolymer-brush-modified glass beads; the measurements were performed using a previously reported method,⁵⁰ with PB as the liquid phase (Fig. 3A). Typical photographs used for contact angle measurements are shown in Fig. 3B. The cos θ value for the PAMPS-b-PIPAAm brushes was lower than that for the PAMPS brushes, and the same as that for the PIPAAm brushes. Also, cos θ for the PAMPS-b-PIPAAm brushes decreased with increasing temperature; the profile for the PIPAAm brushes was similar. These results for the surface wettability of the PAMPS-b-PIPAAm brushes are reasonable. The wettability of the PAMPS brushes would be higher than that of the PIPAAm brushes because of the acidic properties of sulfonic acid groups. Grafting of PIPAAm on PAMPS brushes therefore decreased the cos θ value, i.e., the wettability decreased. Also, the upper segment of the PAMPS-b-PIPAAm brushes was PIPAAm, therefore the surface wettability of block copolymer showed the same profile as that of PIPAAm.

Fig. 3 Contact angles of prepared copolymer-brush-modified glass beads at various temperatures (A), and microscopic image of copolymer-modified glass beads at liquid–air boundary layer (B) (abbreviations are defined in Table 1).

The anionic properties of the copolymer-brush-modified silica beads were investigated by measuring the zeta potentials of the beads at various temperatures (Fig. 4). At low temperatures, the zeta potential of the PAMPS-b-PIPAAm brushes (20AS-600IP) was higher than that of PAMPS brushes (20AS). The zeta potential of 20AS-600IP decreased with increasing temperature and was the same as that of 20AS at high temperatures. This is because swelling of the PIPAAm segment at low temperatures prevented exposure of the anionic charge of the PAMPS base layer. The PIPAAm segment of PAMPS-b-PIPAAm shrank with increasing temperature, and the anionic charge of PAMPS was exposed. The zeta potential of PAMPS-b-PIPAAm therefore decreased with increasing temperature.

Fig. 4 Zeta potentials of copolymer-modified silica beads at various temperatures (abbreviations are defined in Table 1).

Interactions between copolymer brushes and biomolecules

The acidic properties of the copolymer brushes were investigated by performing chromatographic analyses using copolymer-brush-modified beads as the stationary phase and basic catecholamine derivatives as analytes. We tried to investigate the properties of all the prepared copolymer-brush-modified beads. However, 10AS-300IP and 20AS-300IP were not suitable as thermoresponsive chromatography matrices because their PIPAAm segments are short and thermally modulated properties were not observed. However, block copolymer brushes with long PIPAAm brushes, e.g., 10AS-1000IP and 20AS-1000IP, cannot be used as packing materials because long copolymers on silica bead surfaces lead to an excessively high mobile phase flow pressure. We therefore used 10AS-600IP and 20AS-600IP, which have intermediate PIPAAm brush segments, as chromatographic packing materials. Fig. 5 shows the elution behaviors of catecholamine derivatives at various temperatures on columns packed with the prepared beads. Fig. 6 shows the temperature-dependent retention times of the catecholamines. We examined the elution behaviors of the catecholamines at 5 °C intervals from 10 to 50 °C. The chromatograms recorded at 10, 30, and 50 °C, i.e., at equal temperature intervals within the range, are displayed. The elution profiles show that the 10AS-600IP, 20AS-600IP, and 600IP-r-20AS columns retained the catecholamine derivatives, but the 600IP column did not. The results indicate that the catecholamine derivatives were retained by electrostatic interactions with acidic AMPS moieties on the copolymer. The catecholamine retention times on the 20AS-600IP column were longer than those on 10AS-600IP. This indicates that the catecholamine molecules interacted with the base PAMPS layer of the PAMPS-b-PIPAAm brushes, and that the longer PAMPS segment of 20AS-600IP resulted in stronger electrostatic interactions with catecholamines than in the case of 10AS-600IP. The catecholamine retention times on 600IP-r-20AS were similar to those on 20AS-600IP. This indicates that the strengths of the electrostatic interactions between 600IP-r-20AS, which was prepared by random copolymerization, and the catecholamines were similar to those between the catecholamines and 20AS-600IP. The retention times of the catecholamine derivatives on the 10AS-600IP and 20AS-600IP columns increased at around 30 °C, near the lower critical solution temperature (LCST) of PIPAAm. There are two probable reasons for this. One is hydrophobic interactions between the catecholamines and PIPAAm segment in the block copolymer. Our previous study indicated that catecholamine molecules are retarded by both electrostatic and hydrophobic interactions.³⁴ The catecholamine derivatives therefore interacted hydrophobically with the PIPAAm segment, and the hydrophobic interactions were enhanced near the PIPAAm LCST. The other is collapse of the PIPAAm segment of PAMPS-b-PIPAAm with increasing temperature. The base PAMPS segment was therefore exposed at the outer surfaces at high temperatures, leading to enhanced electrostatic interactions with the catecholamines.

Fig. 5 Chromatograms of catecholamines separated by HPLC at various temperatures using copolymer-brush-grafted silica beads as column-packing materials: (A) 10AS-600IP, (B) 20AS-600IP, (C) 600IP-r-20AS, and (D) 600IP (abbreviations are defined in Table 1); mobile phase was 66.7 mmol L⁻¹ PB (pH 7.0). Peak no. 1, DOPA; no. 2, adrenaline; and no. 3, tyramine.

Fig. 6 Changes with temperature in retention times of catecholamines on (A) 10AS-600IP, (B) 20AS-600IP, (C) 600IP-r-20AS, and (D) 600IP (abbreviations are defined in Table 1).

Hydrophobic interactions with steroids were also investigated at various temperatures. Fig. 7 shows chromatograms of steroids at various temperatures, and Fig. 8 shows the temperature-dependent retention times of the steroids. The retention times of the steroids on 10AS-600IP and 20AS-600IP decreased with increasing temperature, but the hydrophobic interactions of 600IP-r-20AS and 600IP increased with increasing temperature. Previous reports have suggested that the retention times of steroids on columns packed with PIPAAm-modified beads increased with increasing temperature.^20,22 The retention profiles on 600IP-r-20AS and 600IP are therefore reasonable. The reasons for the decreased retention times of steroids on 10AS-600IP and 20AS-600IP are as follows. At low temperatures, the retention times of steroids on 10AS-600IP and 20AS-600IP were longer and the chromatogram peaks were broader. The PAMPS-b-PIPAAm brushes were highly hydrated at low temperatures because of the strong hydrophilicity of the PAMPS brushes and hydration of the PIPAAm segment. Steroid molecules therefore diffused into the copolymer brushes, leading to broadening of the steroid peaks at low temperatures. The PIPAAm segment in the block copolymer shrank with increasing temperature, therefore the steroid molecules did not diffuse into the block copolymer brushes and the peaks became narrower. The solubilities of the steroids in the mobile phase also increased with increasing temperature. These factors result in decreased steroid retention times with increasing temperature. The PAMPS-b-PIPAAm brush contact angle measurements show that the hydrophobicity increased with increasing temperature (Fig. 3). However, the steroid molecules interacted with the inner PAMPS-b-PIPAAm brush layer, but the contact angle measurements reflect the hydrophobic properties of the outer surfaces of the brushes. The contact angle measurements and chromatographic analysis therefore gave different results for the hydrophobic properties.

Fig. 7 Chromatograms of steroids separated by HPLC at various temperatures using copolymer-brush-grafted silica beads as column-packing materials: (A) 10AS-600IP, (B) 20AS-600IP, (C) 600IP-r-20AS, and (D) 600IP (abbreviations are defined in Table 1); mobile phase was 66.7 mM PB (pH 7.0). Peak no. 1 hydrocortisone and no. 2 dexamethasone.

Fig. 8 Changes with temperature in retention times of hydrophobic steroids on (A) 10AS-600IP, (B) 20AS-600IP, (C) 600IP-r-20AS, and (D) 600IP (abbreviations are defined in Table 1).

Protein adsorption profiles on block copolymer brushes

We examined the adsorption profiles on the PAMPS-b-PIPAAm brushes of proteins with various basic properties and molecular weights. Protein molecules are larger than catecholamine and steroid molecules, therefore the adsorption profiles of proteins provide different information on the copolymer brushes. Fig. 9 shows typical protein chromatograms, and Fig. 10 shows the protein adsorption profiles. The chromatograms recorded at 10 and 40 °C are displayed in Fig. 9; the elution profiles at these temperatures clearly show protein elution and adsorption. The peak areas of several proteins decreased significantly with increasing temperature on 10AS-600IP, 20AS-600IP, and 600IP-r-20AS columns, but only decreased slightly on the 600IP column. The chromatograms show that α-chymotrypsinogen A was not eluted from the 20AS-600IP column at 40 °C, but was eluted at 10 °C (Fig. 9A). This indicates that α-chymotrypsinogen A was adsorbed on the copolymer brushes at high temperatures but not at lower temperatures. A possible protein adsorption mechanism is as follows. At low temperatures, the PIPAAm segment of PAMPS-b-PIPAAm is hydrated and swollen, therefore α-chymotrypsinogen A cannot access the base PAMPS layer of PAMPS-b-PIPAAm and is not adsorbed. In contrast, at high temperatures, α-chymotrypsinogen A can access the PAMPS segment and is adsorbed on the copolymer brushes; this is because the PIPAAm segment is dehydrated and shrunken, leading to enhanced electrostatic and hydrophobic interactions. The smaller peak area shows that adsorption of α-chymotrypsinogen A was stronger on 20AS-600IP than on 10AS-600IP, indicating that the PAMPS segment in 20AS-600IP is longer than that in 10AS-600IP, resulting in stronger electrostatic interactions with acidic α-chymotrypsinogen A (Fig. 10). α-Chymotrypsinogen A was not adsorbed on the 600IP-r-20AS column (Fig. 9). This shows that random copolymer brushes with widely distributed ionic groups were not effective for protein adsorption, unlike block copolymer brushes. Lysozyme was adsorbed at all temperatures on the 10AS-600IP and 20AS-600IP columns (Fig. 10). This is probably because lysozyme molecules are smaller than α-chymotrypsinogen A molecules (Table S2†). Lysozyme penetrates the copolymer brushes, and can interact with the acidic PAMPS base layer although the PIPAAm segment in the block copolymer swells at low temperatures. At high temperatures, lysozyme is adsorbed on the copolymer brushes through electrostatic interaction with the exposed sulfonic acid groups in the base PAMPS layer and hydrophobic interactions with dehydrated PIPAAm. These factors lead to strong adsorption of lysozyme on the PAMPS-b-PIPAAm brushes. In contrast, temperature-dependent adsorption of lysozyme on the 600IP-r-20AS column was observed. The reasons for this adsorption profile are as follows. At low temperatures, lysozyme diffuses into the copolymer brushes and interacts with random copolymer brushes through electrostatic interactions with the sulfonic acid groups distributed throughout the copolymer. The lysozyme peak is therefore broad at low temperatures (Fig. 9D). Lysozyme adsorption on the copolymer brushes increases with increasing temperature because the copolymer becomes dehydrated and shrunken, leading to exposure of the widely distributed sulfonic acid groups of AMPS at the outer surfaces. Lysozyme adsorption on 600IP-r-20AS is therefore temperature dependent.

Fig. 9 Temperature-dependent elution of basic proteins from 20AS-600IP (A, C, and E) and 600IP-r-20AS (B, D, and F); (A) and (B) α-chymotrypsinogen A, (C) and (D) lysozyme, and (E) and (F) trypsinogen; mobile phase was 66.7 mM PB (pH 7.0) (abbreviations are defined in Table 1).

Fig. 10 Changes with temperature in peak areas of proteins eluted from columns packed with prepared beads: (A) 10AS-600IP, (B) 20AS-600IP, (C) 600IP-r-20AS, and (D) 600IP (abbreviations are defined in Table 1).

The adsorption profiles of α-chymotrypsinogen A and lysozyme show that 20AS-600IP, which was prepared by block copolymerization, has strong protein adsorption properties as a result of thermally modulated exposure of the PAMPS base layer. The block copolymer brushes could therefore be used as protein adsorption materials, with properties that can be changed by changing the external temperature.

Conclusions

Silica bead surfaces were modified with brushes of a block copolymer, namely PAMPS-b-PIPAAm, with strong anionic properties and a thermoresponsive segment, using two-step surface-initiated ATRP. XPS, CHN elemental analyses, and GPC measurements showed successful grafting of PAMPS and PIPAAm. Contact angle measurements performed on copolymer-brush-modified glass beads showed that the surface wettability of PAMPS-b-PIPAAm is similar to that of PIPAAm, indicating that the surface wettability depends on the properties of the upper segment of the copolymer brushes. Zeta potential measurements showed that the anionic properties of the PAMPS-b-PIPAAm brushes strengthened with increasing temperature because of shrinkage of the PIPAAm segment and exposure of the PAMPS layer. Chromatographic analyses were performed using the copolymer-modified beads as column-packing materials. The elution profiles showed that the retention times of catecholamines were longer on PAMPS-b-PIPAAm brushes with long PAMPS segments. This indicates that small basic molecules interact with the base PAMPS brush layer. Basic protein elution profiles indicated that the PAMPS-b-PIPAAm brushes are a stronger adsorbent than brushes of the random copolymer P(IPAAm-co-AMPS); this is because of effective thermally modulated exposure of the PAMPS brushes. PAMPS-b-PIPAAm brushes could be used as intelligent interfaces with thermally modulated variations in the surface anionic properties.

Acknowledgements

This research was partly financially supported by the Development of New Environmental Technology Using Nanotechnology Project of the National Institute of Environmental Science (NIES), commissioned by the Ministry of the Environment, Japan; Grants-in-Aid for Scientific Research (C) No. 26420714 from the Japan Society for the Promotion of Science (JSPS), Japan; and subsidies from the Kumagai Foundation for Science and Technology.

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Footnote

† Electronic supplementary information (ESI) available: Properties of catecholamine, steroids, and proteins, deconvolution of C 1s XPS peaks, gel-permeation chromatograms of copolymers. See DOI: 10.1039/c6ra20944k

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