Surface modified microfiltration membranes with molecularly recognising properties

Journal of Membrane Science 213 (2003) 97–113

Surface modified microfiltration membranes with molecularly recognising properties Nidal Hilal a,∗ , Victor Kochkodan b a

b

School of Chemical, Environmental and Mining Engineering, University of Nottingham, University Park, Nottingham NG7 2RD, UK Institute of Colloid and Water Chemistry, National Academy of Science of Ukraine, Vernadskii Pr. 42, 03142 Kiev, Ukraine Received 27 June 2002; received in revised form 22 October 2002; accepted 24 October 2002

Abstract Polyvinilidene fluoride (PVDF-phob and PVDF-phil) and polyethersulfone (PES) microfiltration membranes were surface modified with a thin layer of molecular imprinted polymer (MIP). This material is selective to adenosine 3:5-cyclic monophosphate (cAMP) via photoinitiated copolymerisation of 2-(dimethylamino)ethyl methacrylate as a functional monomer and trimethylopropane trimethacrylate as a cross-linker in the presence of cAMP in ethanol/water solutions. The specific and non-specific template binding of MIP during filtration of aqueous solutions of cAMP was studied for membranes with different degrees of modification. It was concluded that the ability of MIP membranes to bind cAMP is a result of both the specific size and shape of recognising sites in addition to the correct position of the functional groups involved in the template binding through ionic and hydrogen binding interactions. Profile imaging atomic force microscopy and scanning electron microscopy were used to visualise surfaces and cross-sections of MIP membranes. The main advantages of this approach for MIP membrane preparation are very fast MIP layer synthesis and the possibility to obtain MIP composite membranes by controlled deposition on different kind of polymeric supports. Atomic force microscopy in conjunction with the coated colloid probe technique has been used to measure interactions between a silica sphere coated with imprinted polymer and porous supports. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Molecularly imprinted membranes; Molecular recognition; Atomic force microscopy; Colloid probe; Scanning electron microscopy

1. Introduction Over the last decade methods of developing membranes with controlled specificity for individual compounds have attracted considerable attention [1,2]; for example, selective separation can be performed with affinity membranes using typically a biological receptor, e.g. an antibody, immobilised on a suitable ∗ Corresponding author. Tel.: +44-115-9514168; fax: +44-115-9514115. E-mail address: [email protected] (N. Hilal).

membrane [1]. The main limitation of such an approach is the lack of suitable receptors for many compounds, their low stability and/or high cost. The use of molecular imprinted polymers (MIPs) for developing membranes capable of recognising individual compounds seems to be an attractive option [3]. Molecular imprinted polymers are synthetic biomimetic materials that posses affinity and selectivity toward templates due to special recognition sites formed during the preparation of a polymeric matrix [4,5]. The preparation of MIPs consists of copolymerisation of functional and cross-linker monomer

0376-7388/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 6 - 7 3 8 8 ( 0 2 ) 0 0 5 1 6 - 1

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in the presence of a template molecule. Subsequent removal of the template molecules leaves behind receptor sites, which are complementary to the template in shape and position of the functional groups. In this way a molecular memory is introduced into the polymer, which becomes capable of selectively rebinding the template molecule [4–9]. The polymeric nature of MIPs results in advantages over natural biological receptors in terms of physical and chemical stability and the possibility of using MIPs in harsh environmental conditions. Two approaches, covalent and non-covalent, have been developed for molecular imprinting [4,5]. In the former, polymerisable derivative of the template is synthesised and used as a template. The subsequent extraction of such a template requires cleavage of covalent bonds. In the latter, a template interacts with both a functional monomer and MIP via non-covalent interaction, e.g. ionic, hydrophobic and hydrogen bonding. The non-covalent approach seems to be more flexible towards the choice of functional monomers and possible template molecules and has therefore been more widely used [5–8]. Usually MIPs are produced by bulk polymerisation of a mixture of functional monomers and template, followed by grinding of the obtained polymer to particles. MIPs have been widely used in studies on chromatography separation [6,8], receptor mimics in assay systems [9], recognition elements in biological sensors [3,10] and artificial enzyme catalytic systems [11]. However, the practical application of MIPs is still under development [12]. It has been shown in the last decade that membranes prepared by means of the phase inversion technique in the presence of a template molecule possess molecular recognition properties [13–16]. Surface modification by grafting in the presence of a template can be also used for synthesis of MIP membranes [17,18]. It has been reported recently that composite thin layer MIP membranes can be prepared using a ␣-cleavage photoinitiator for initiation of copolymerisation reactions in a monomer mixture [19]. In the present work this approach has been extended to prepare selective MIP layers on the surface of various microfiltration membranes using adenosine 3:5-cyclomonophosphate (cAMP) as a template. This chemical is one of the most important second messengers in cells, acting as an intracellular regulator, which is also involved in regulating neuronal, glandular,

cardiovascular, immune and other functions [20]. Measurement of cAMP concentration is important for studies on numerous hormones, local mediators, neurotransmitters, drugs and toxins. Biological recognition elements, such as antibodies, are typically used to bind cAMP. This is an expensive procedure, which requires special techniques [21]. Another highly desirable approach is using artificial synthetic systems for cAMP recognition, including the imprinting of membranes. Imprinting of this cAMP template if successful should be performed from a polar medium since it is insoluble in organic solvents. This is of special interest because so far non-covalent molecular imprinting protocols have been mainly limited to non-polar organic solvents [7,12]. To gain better understanding of the MIP layer deposited on membranes, atomic force microscopy (AFM) and scanning electron microscopy (SEM) were used in this work to visualise surfaces and cross-sections of MIP membranes. Atomic force microscopy produces three-dimensional topographical images by scanning a sharp ultralever tip over a surface [22]. The key advantages of AFM over other techniques are its ability to produce high-resolution images of membrane surfaces without special sample preparation [23–25]. Recent developments in the use of AFM make possible the visualisation of surface down to the nanometer resolution [26] and the direct measurement of the force of adhesion of a coated colloid probe to a membrane surface [27]. In this work we exploit AFM capabilities to perform a surface morphology analysis of MIP membranes and to directly quantify the forces of adhesion between coated colloid probe with imprinted polymer and different types of polymer membranes used as porous support for synthesis of composite MIP membranes.

2. Materials and methods Hydrophobic and hydrophilic polyvinylidene fluoride (PVDF-phob and PVDF-phil, Durapore) and polyethersulfone (PES, Millipore Express), microfiltration membranes were used in this study as porous supports for preparation of composite MIP membranes. Benzoin ethyl ether (BEE), trimethylopropane trimethacrylate (TRIM), 2-(dimethylamino)ethyl

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methacrylate (DMAEM), 2-hydroxyethyl methacrylate (HEMA), methacrylic acid (MA) and adenosine 3:5-cyclic monophosphate (cAMP) were obtained from Sigma–Aldrich. The inhibitor was removed from the monomers by passing them through a column of aluminium oxide (activated basic, Brockmann, Sigma–Aldrich) immediately before use. 2.1. Synthesis of composite MIP membranes To obtain the imprinted polymer layer on the surface of initial membranes, the samples were coated with photoinitiator by soaking in 0.25 M BEE in methanol for 2–3 min. After drying at 40 ◦ C the samples were immersed in a solution containing 3 mM of cAMP, 50 mM of DMAEM as a functional monomer and 200 mM of TRIM as a cross-linker in ethanol/water mixture (70/30 (v/v)). Samples were UV irradiated with a B-100 lamp (Ultra-Violet Products Ltd.) with a relative radiation intensity of 21.7 mW/cm2 at a wavelength of 350 nm. MIP membranes with different degrees of modification (DM) were obtained by varying the time of UV exposure (polymerisation time). After polymerisation, membranes were extracted with an ethanol/water solution for at least 4 h and consistently washed with 0.1 M NaOH and distilled water to remove any non-grafted polymer, monomer, residual initiator and the template. The efficiency of this procedure was checked by filtration of the distilled water through a membrane and recording the UV absorbance of the filtrate. The absorbance was less than 0.005 at 258 nm. The degree of modification was calculated from the difference in weight between the modified sample with the deposited MIP layer and the initial unmodified sample. The reproducibility of DM values was found to be less than 10%. For comparison, blank membranes (without template) were prepared under the same conditions. Attenuated total reflection/Fourier transform infrared (ATR/FTIR) spectra of initial and MIP-modified membranes were determined using a Protégé 460 FTIR spectrometer (Nicolet Instrumental Corp.). One hundred scans were obtained at a resolution of ±2 cm−1 . A ZnSe internal reflection element was used in the ATR at an incident angle of 45◦ , giving an IR penetration depth of 0.42–4.2 ␮m.

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2.2. Filtration experiments A UF stirrer cell of 25 mm diameter and volume of 10 cm3 (Amicon, Millipore) was used for measuring membranes water fluxes at different operating pressures. Binding of cAMP with MIP membranes was evaluated from filtration tests. A 5 × 10−5 M solution of cAMP in water was filtered through the membrane at a rate of 1 ml/min using a syringe connected to a 25 mm filter-holder (Swinnex, Millipore). Sorption values were calculated from the difference of the template concentrations in the feed and permeate, respectively. A UV spectrophotometer (DMS 80/90, Varian Techtron, Australia) was used for quantitative determination of cAMP (λ = 258 nm). 2.3. Surface morphology and cross-section study by AFM and SEM Atomic force microscopy and scanning electron microscopy were used to visualise the surface structures and cross-section of both the initial and modified membranes. The AFM used in this study was an Explorer (TMX 2000), a commercial device from Vecco Instruments (USA). A silicon cantilever (Ultralevers, Park Scientific Instruments) with a high aspect ratio tip of typical radius of curvature 5–10 nm was used to scan the membrane and produce the images. On the AFM, profile imaging mode was selected to study the polymeric membranes at a temperature of 25 ◦ C. This imaging mode has not been previously used for imaging membrane surfaces; it has many advantages over other AFM modes. In the profile imaging mode, the image was acquired by having the tip approach the surface at a speed of 300 ␮m/s, at such speed the feed back signal was more positive than the set point, −5 nA. The Z-level was recorded as topographic information. The tip was retracted above the sample by a fixed distance (Z-pullout of 3100 nm) and the tip was moved laterally to the next position at the top of the pullout cycle. The process was repeated at each data point to establish the topographic profile of the membrane surface. The images were obtained over an area of 30 ␮m × 30 ␮m for both initial and modified membranes. Membrane surface parameters such as the root mean square of Z-values and total contact area can be obtained from the images [23,28].

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The root mean square of Z-values (RMS) is defined as the square root of the mean value of the triangles of the distance of the point from the image mean value over each data point:
 N 1 RMS =  Zi − Z 2 (1) N

where Z12 = Z1 − Z2 .  b = (y)2 + (Z24 )2

(4)

capabilities that have proven essential for the effective study of membrane surfaces. Line analysis and surface statistics such as surface roughness, average height, and maximum peak-to-valley distance may be collected. The simultaneous use of images and profiles greatly facilitates identification of the entrance of individual pores. Pore diameters were determined in this way for 25 pores. Scanning electron microscopy (SEM) was used in this study to produce cross-sectional images of initial PVDF and MIP membranes with different degrees of modification. An ISI-SX-30 scanning electron microscope (operating at 30 kV electron accelerating voltage) was employed to view the cross-section of the membrane samples. All photomicrographs were obtained using secondary electron detection mode. In order to prepare a clean edge for the SEM imaging, each membrane was broken under liquid nitrogen. Each membrane was allowed to cool in the nitrogen for 2–3 min before breaking. Care was taken to avoid creating compression or stress near the break point. Each sample was vertically attached with the aid of tweezers onto a 30 mm diameter aluminium stub using silver dag paste. Once dry each sample was then coated for 80 s with a fine layer of gold particles (∼250 Å) using Polaron gold coating equipment. Images were taken at magnifications between 380 and 8000 times.

where Z24 = Z2 − Z4 .  c = (z)2 + (Z14 )2

(5)

2.4. Measurement of adhesion interaction using AFM colloid probe technique

i=1

where Zi is the current Z-value, while Z and N are the average of Z-values and the number of points within the area, respectively. Average height gives the arithmetic mean defined as: N

1 |Z| = Zi N

(2)

i=1

Total contact surface area is calculated including the height (Z-data: Z1 , Z2 , Z3 and Z4 ) of every four adjacent pixels. The surface of the square described as Z1 , Z2 , Z3 and Z4 is computed by dissecting the square into triangles and then computing the area of each triangle.  (3) a = (x)2 + (Z12 )2

where Z14 = Z1 − Z4 . The surface of a single triangle is then calculated as:  S = p(p − a)(p − b)(p − c) (6) where p = (1/2)(a + b + c). This calculation was applied to the entire imaged surface in order to get the total surface area including the Z-variations. So the ratio of the projected area over the surface area gives a good indication of the sample surface area deviation in relation to the geometric flat surface. Once the image of membrane surface is taken, the software supplied with the Explorer (TMX 2000) stores the picture as a data matrix of co-ordinates enabling us to measure pore diameter using digitally stored line profiles. The Explorer has image analysis

To quantify the adhesion interactions between the colloid probe coated with MIP and the polymer support, the force between a cantilever and a membrane sample as a function of the scanner displacement (the latter being varied using a piezoelectric crystal) was measured. A laser beam reflected from the back of the cantilever falls onto a photosensitive position detector (PSPD) photodiode which detects changes in cantilever deflection. To generate a force–distance curve, the deflection of the cantilever tip is recorded as a function of the tip-sample separation as the piezo scanner raises the sample towards the tip and retracts away. To convert the data into force as a function of true sample-tip separation, it is necessary to know the spring constant of the cantilever and to define zero values of both force and separation distance [29].

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The geometry of AFM cantilever tips is often unknown therefore it was replaced by replaced by a sphere of known geometry in the present work creating a colloid probe. The probe was made by attaching a 5 ␮m diameter silica sphere (Polysciences Inc.) to a tipples ultralever-type cantilever (Park Scientific Instruments, USA) whose spring constant K was 0.05 N/m (the mass of the sphere is not great enough to significantly modify the spring constant of the bare cantilever). This was in good agreement with that calculated from the dimensions of the cantilever [30]. Colloid spheres of this type have been shown to have a maximum peak-to-valley roughness of 3 nm over an area of 0.45 ␮m2 [29]. The silica colloid probe was coated with the imprinted polymer layer using the same procedure as for preparation MIP membrane samples (Section 2.1). Prior to coating the colloid probe was rinsed with ethanol and flushed copiously with deionised water. Loading force on the cantilever was found to have a significant effect on the value of adhesion force [31]. Therefore, all force measurements were conducted at constant loading force.

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Benzoin ethyl ether (BEE) a ␣-scission type photoinitiator was used in this work to generate the copolymerisation reaction in the monomer mixture and obtain a thin layer of MIP on the surface of polymer supports. The cleavage of the BEE photoinitiator that creates starting radicals for copolymerisation in the monomer mixture is shown below [32].

the photoinitiator concentration from the membrane surface. It can be assumed that the highest concentration of starter radicals, generated by the UV radiation, is located close to the membrane surface. As the membrane surface is practically inert to the generated radicals, the modification may be considered as a deposition of MIP layer on the surface of polymer support [19]. The degree of modification (DM) of support membranes depends on the loading concentration of photoinitiator during pre-soaking as well as the time of polymerisation. Modification of polymer supports is a very fast process under these conditions. Even the highest degrees of DM were obtained after short durations of UV exposure as shown in Fig. 1 for PES, PVDF-phil and PVDF-phob membranes. Methacrylate-based polymers have been most widely used in non-covalent imprinting protocols [4–8]. Several functional monomers, DEAEM, HEMA and MA at different concentrations of the functional monomer (20–130 mM) were used in this work for screening experiments to optimise the composition for MIP membrane preparation. It was found that DEAEM gave MIP membranes the best binding with cAMP. A gradual increase of TRIM concentration in the monomer mixture (up to 200 mM) was found to increase cAMP sorption, this is most probably due to improved stabilisation of the structure of selective cavity. Further increase in TRIM concentration in the reaction mixture (>200 mM) led to a decrease of cAMP sorption, this may be due to formation of an increasing fraction of excessively cross-linked domains in MIP matrix. These domains possess a reduced number of MIP receptor sites as well as poor accesses to receptor sites for the template. Fig. 2 shows the effect of DMAEM concentration on sorption of cAMP in both MIP and blank membranes.

In contrast to benzophenone, which creates starting radical for photoinitiated surface grafting by hydrogen-abstraction from the polymer backbone, ␣-cleavage photoinitiator had no effect on polymer support. Immersing BEE coated membrane samples in monomer/template solutions creates a gradient of

It may be seen in Fig. 2 (curve 1), that an increase of DMEAM concentration in the reaction mixture leads to an increase of cAMP sorption with MIP membranes. The increase was sharp between 20 and 50 mM of DMAEM concentration, followed by a gradual increase between 50 and 130 mM. This is most probably

3. Results and discussion 3.1. Preparation and characterisation of composite MIP membranes

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Fig. 1. Effect of UV exposure time on modification of microfiltration membranes with MIP layer (loading of BEE 2.5 mg).

Fig. 2. Effect of DAEAM concentration on sorption of cAMP on MIP and blank PVDF-phob membranes (DM of 820 ± 20 ␮g/cm2 ).

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due to the creation of an increasing number of specific template binding sites in the imprinted polymer. The non-specific sorption of cAMP on blank membrane samples also tends to increase with DMEAM concentration; in this case the increase was gradual over the whole range of concentration (Fig. 2, curve 2). The specificity of membranes, which reflects the maximal imprinting effect, was calculated from the difference of sorption between MIP and blank samples and shown as curve 3 in Fig. 2. As a result the optimal value for DEAEM concentration in the reaction mixture was found to be 50 mM (Fig. 2, curve 3). Attenuated total reflection/Fourier transform infrared (FTIR/ATR) technique was used to confirm that deposition of new MIP layer on the surface of initial membranes had taken place. The ATR/FTIR spectra of initial and MIP-modified PVDF-phob membranes are shown in Fig. 3. This figure shows that the most significant change in the spectra of modified MIP membranes is the identification of a carbonyl band at ∼1728 cm−1 as well as bands at 2850 and 2930 cm−1 corresponding to methylene groups. All these bands are not present in the case of initial PVDF-phob membranes. The appearance of these bands confirms that a new molecular imprinted copolymer layer was deposited on the surface of the polymer support. 3.2. Template affinity and imprinting effect The template affinity to MIP and blank membranes was evaluated from filtration experiments using 5 × 10−5 M aqueous solution of cAMP. Both MIP and blank modified membranes showed enhanced cAMP sorption compared to initial membranes, whose sorption was less than 4%. Difference in sorption between MIP and blank samples prepared under identical conditions (specificity of MIP membranes) gives information regarding specific binding to recognising sites allowing the imprinting effect to be evaluated. The effect of degree of modification on the specificity of membranes is shown in Fig. 4. This figure shows that the specificity of membranes increases sharply with an increase of degrees of DM to a maximum value followed by a decrease. The increase of DM most probably leads to an increase of the thickness of the MIP layer. The thicker the deposited imprinted layer, the larger the number of recognition sites that are available for the template molecules when passing through the

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membrane. The largest imprinting effect was observed for MIP membranes at DM value of 920 ␮g/cm2 as shown in Fig. 4. Further increase of degrees of modification gave a reduction in the specificity of membranes. This finding may be ascribed to a gradient in BEE concentration in the monomer mixture nearby the surface of the sample coated with photoinitiator. Such a gradient leads to alteration of the density of the created polymer network in a direction away from the membrane surface. As a result, the thickness of the imprinted polymer layer with optimal recognising properties is limited. Aqueous solutions of 5 × 10−5 M cAMP used for the filtration experiments have a pH 4.2. Dimethylamino groups are positively charged at this pH [33]. It can be assumed that the ionic interactions between the phosphorous residue in the cAMP molecule and the protonated dimethylamino groups of MIP network contribute to the affinity of MIP membranes. This was proved by a decline of cAMP sorption when the ionic strength of the solution is increased as shown in Table 1. The ionic interaction becomes weaker with increasing NaCl concentration due to more effective shielding of charged groups. It also shows that the sorption of cAMP is sensitive to the pH of the filtered solution. This is probably due to the change in degree of protonation of the dimethylamino groups [34]. Table 2 shows sorption of adenosine 3:5-cyclic monophosphate (cAMP) and quanosine 3:5-guanidine cyclic monophosphate (cGMP) on MIP membranes with different degree of modification. It may be seen from this table that essentially less sorption of structurally similar cGMP is obtained for MIP membranes compared to cAMP. Both cAMP and cGMP molecules include a phosphorous group and they only differ in the substituents on their purine base moiety. The adenine base of cAMP contains an NH2 group

Table 1 Influence of pH and NaCl concentration on the cAMP sorption on MIP and blank PVDF-phob membranes at DM of 1100±40 ␮g/cm2 Membrane

MIP Blank

NaCl concentration (M)

pH

10−4

10−3

10−1

4.2

11.2

70 24

42 10

32 8

72 24

22 6

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Fig. 3. FTIR/ATR spectra of the surface of (a) initial and (b) MIP-modified PVDF-phob membranes.

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Fig. 4. Specificity of MIP-modified PVDF-phob membranes at different degrees of DM.

Table 2 Sorption of cAMP and cGMP and binding capacity of MIPmodified PVDF-phob membranes at different degrees of modification Degree of modification (±20 ␮g/cm2 )

Sorption (%) cAMP

cGMP

0 540 920 1100

4 44 67 72

3 22 30 41

Binding capacity to cAMP (␮g/cm2 )

4.2 10.5 12.6

at the C6 position, whereas in the guanine base of cGMP the same position is replaced with a carboxyl group, the C2 position is substituted with an NH2 group as shown in the formula below.

The ability of the cAMP imprinted membranes to distinguish between cAMP and the structurally similar cGMP suggests that the binding of cAMP to the recognising sites is not only based on ionic interactions. Other interactions may also contribute to affinity of MIP membranes, possibly hydrogen bonding between amino and hydroxyl groups of template molecule and carbonyl groups of MIP. The size and the shape of the binding cavity along with the correct spatial orientation of the functional groups in the MIP binding sites also play an important role [4,6,10]. Thus, molecular recognition of cAMP may be attributed both to the specific size/shape of recognising sites complementary to the template as well as correct position of the

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functional groups in the cavity involved in template binding. These results indicate the possibility to develop a non-covalent molecular imprinting protocol in polar mediums using ionic and hydrogen bonding interactions between the template and the functional monomer. A schematic representation for the formation of the hypothetical recognition sites specific to cAMP in imprinted polymer matrix is presented in Fig. 5. Fig. 5a shows the arrangement of DMEAM molecules around the cAMP molecule; this results in the formation of pre-polymerisation complex. Preliminary arrangement of functional monomer molecules around a template is assumed to be an important factor for a successful imprinting procedure [5,6]. The structure of this complex is fixed in a rigid polymer matrix created during the polymerisation of the functional monomer with a cross-linker as shown in Fig. 5b. After template extraction the specific recognition sites complementary to the cAMP in the size, shape and in the position of the functional groups are formed in the polymer matrix (Fig. 5c). The MIP is capable of rebinding cAMP molecules from aqueous solutions because of these specific sites. Sorption of cAMP on blank membranes proceeds mainly via non-specific binding with the statistically distributed dimethylamino groups of the polymer matrix. Fig. 6 shows the water fluxes for MIP membranes at various degrees of modification versus operating pressure. It may be seen from this figure that the fluxes for MIP membranes were lower than the flux of the initial membrane. However, they were still high enough and well suited for fast membrane solid-phase extraction. The total binding capacities for MIP membranes were determined by consecutive filtrations of 3 ml fractions of cAMP solution. The data are summarised in Table 2. It may be seen from this table that the total binding capacity of MIP membrane increases with increasing DM. The binding capacity of cAMP was saturated at relatively low permeate volumes (12–15 ml)

䉴 Fig. 5. Schematic representation for formation of hypothetical recognising sites in the MIP matrix: (a) formation of prepolymerisation complex between the functional monomer and the template; (b) fixation of the structure of pre-polymerisation complex in MIP matrix during copolymerisation of the functional monomer and the cross-linker; (c) formation of specific binding site via extraction of the template.

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Fig. 6. Water fluxes for MIP membranes with different degrees of modification.

due to the small area and thickness of MIP membrane samples. However, the high MIP membrane water fluxes makes it possible to use these membranes in stacks. MIP membranes may be re-used after regeneration with 0.1 M NaOH and subsequent washing with water to release sorbed cAMP. 3.3. Surface morphology and cross-section of MIP membranes Atomic force microscopy (AFM) and scanning electron microscopy (SEM) were used to visualise surfaces and cross-sections of MIP membranes. Fig. 7 shows high-resolution AFM images of initial and modified PVDF membranes with different degrees of modification. Figures are shown in three-dimensional form over an area of 35 ␮m × 35 ␮m. For the analysis of surface pore characteristics the AFM image processing program was used. This allows work with different colour tables and adjustment of the contrast and image representation in top view and in perspective. In addition, its ability to generate line profiles along selected lines in the images was utilised

for the analysis of single pores and region profiles. The colour density in Fig. 7 shows the vertical profile of the sample with the light regions being the highest points and the darkest regions being the pores. The pores are clearly visible as well-defined dark areas, their pore size was determined using the image processing program. Pore size determination is described in full details elsewhere [23]. As has been expected the pore size of MIP membranes decreases with the increase of DM as may be seen in Table 3. This table shows that modification of porous support with imprinted layer leads to increasing the surface roughness (RMS) and Table 3 AFM surface characteristics of MIP-modified PVDF-phob membranes at various degrees of modification (DM) Degree of modification (±20 ␮g/cm2 )

RMS (nm)

Total contact area (␮m2 )

Mean pore diameter (␮m)

0 540 860 1100

192.3 198.1 199.4 296.4

1020 1103 1116 1167

1.08 0.49 0.45 0.43

± ± ± ±

0.10 0.16 0.15 0.18

108 N. Hilal, V. Kochkodan / Journal of Membrane Science 213 (2003) 97–113 Fig. 7. Three-dimensional AFM images of initial PVDF-phob membrane and MIP membranes with different degree of modification: (a) initial membrane; (b) 540 ␮g/cm2 ; (c) 860 ␮g/cm2 ; (d) 1100 ␮g/cm2 .

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Fig. 8. SEM cross-section images of initial PVDF-phob membrane and MIP membranes with different degree of modification: (a) initial membrane; (b) 540 ␮g/cm2 ; (c) 860 ␮g/cm2 ; (d) 1100 ␮g/cm2 .

an increase in the total contact surface area of MIP membranes. The increase of RMS and contact surface area can also contribute to the rise of cAMP sorption with DM. SEM cross-sectional images for these membranes are presented in Fig. 8 at different degrees of modification. It can be seen from this figure that a very thin imprinted layer is formed on the surface of polymer support. The thickness of the MIP layer tends to increase with DM, but this thickness does not exceed 10 nm even at the highest degree of modification 1100 ␮g/cm2 . The small thickness of imprinted polymer layer preserves high fluxes for MIP membranes. 3.4. Adhesion interaction using AFM colloid probe technique A typical force measurement between the silica probe and initial PES membrane is presented in Fig. 9. This figure shows snap back (PSPD sensor output)

of −9 nA. This is equivalent to an adhesion force of 3.75 mN/m between the silica sphere and PES membrane. A typical silica colloid probe is shown in Fig. 10. Fig. 9 shows AFM force–distance experiments for approach and retraction after the colloid probe has been brought into momentary contact with the membrane surface. When the probe is located far from the surface (‘a’ in Fig. 9) with the cantilever in its equilibrium (undeflected) position for which the corresponding ‘zero-force’ photosensitive position detector (PSPD output is constant. At ‘b’ the cantilever touch the surface ‘snap in’, this is taken as the surface position. The scanner start retracting from the surface at point ‘c’ where cantilever is bent upward, Fig. 9 shows retraction over a specified distance of 1 ␮m where the snap back at ‘d’ is taken as the force of adhesion. The PSPD output is presented as a function of piezo position relative to the piezo’s starting height, not to the planar surface. A change in PSPD output (relative to the zero-force) is produced by a deflection of the

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Fig. 9. Typical force curve obtained for the approach and retraction of silica probe to and from PES membrane surface.

Fig. 10. SEM image of 5 ␮m silica colloid probe immobilised at the apex of AFM ultralever.

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cantilever and this causes an additional vertical displacement of the probe, which is independent of scanner position. The region between ‘b’ and ‘c’ is referred to as the region of constant compliance in which the PSPD output is a linear function of scanner position and the probe is considered to be ‘immobilised’ on the solid surface. It follows that the magnitude of the piezo displacement is equal to the magnitude of displacement of the probe relative to the piezo (corresponding to a compressive deflection of the cantilever). Under such circumstances, the relationship between PSPD output and cantilever deflection is calculated from the gradient of the constant compliance region and this relationship is assumed to be valid for deflections produced by the action of tensile forces on the cantilever [29]. The position of the surface indicated in Fig. 9 is inferred from the force profile and the value of the ‘zero-force’ as follows. The cantilever deflection is measured relative to its equilibrium position (being also relative to the position of the piezo column). The position of the surface may then also be related to the position of the piezo column: this is deemed to be the point at which the cantilever is in contact with the surface and in its equilibrium (undeflected)

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state. This ‘zero-distance’ corresponds to position ‘b’ in Fig. 9, from which the separation distance of the probe from the surface, at any given scanner position was evaluated. Normalised adhesion forces between MIP-coated colloid probe and three initial membranes (PES, PVDF-phob, PVDF-phil) are presented in Fig. 11. Experiments were carried out between the MIP-coated colloid probe and membranes at different locations on each membrane surface. Fig. 11 shows that adhesion between the coated probe and PVDF-phob in the range of 0.04–0.082 mN/m, PVDF-phil is between 0.68 and 0.135 mN/m, and PES is between 0.14 and 0.18 mN/m. It was clear from experiments that the MIP-coated colloid probe has weaker adhesion with PES initial membrane (0.14–0.18 mN/m) compared to the value measured between the silica probe and initial PES membrane (3.75 mN/m). It may be seen in Fig. 11 that the force of adhesion between MIP-coated probe and porous support is higher for hydrophilic (PES, PVDF-phil) compared to hydrophobic (PVDF-phob) membranes. This is most probably due to the presence of polar amino and carbonyl groups in the imprinting polymer. The introduced polar groups can increase the interfacial attraction through specific

Fig. 11. Adhesion between MIP-coated colloid probe and different porous polymer supports.

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interaction [35]. Comparison of adhesion results is shown in Fig. 11 with modification data shown in Fig. 1 indicates that a correlation may be found between the degree of modification of the polymer supports and the adhesive force. This correlation is especially noticeable at short polymerisation times (0.5–3 min), when the formation of MIP layer on the support surface has started. The degree of modification is the highest for PES membranes followed by PVDF-phil and PVDF-phob (Fig. 1). This finding was similar for adhesion force (Fig. 11). The influence of porous support becomes weaker with increasing polymerisation time and thickness of MIP layer.

4. Conclusions This paper has shown that composite imprinted membranes with molecularly recognising properties can be developed via photoinitiated synthesis of MIP on the surface of porous polymer supports using a ␣-cleavage photoinitiator. The main advantages of this approach for MIP membrane preparation are very fast MIP layer synthesis and the possibility to obtain MIP composite membranes by controlled deposition on different kind of polymeric supports. It has been found that the adhesive force between a colloid probe coated with MIP and porous polymer support correlates well with the degree of modification at short polymerisation times. The specificity of MIP membranes has also been found to increase with degrees of DM to a maximum value followed by a decrease. Thus, the maximum imprinting effect does not correspond to the highest degree of modification, most probably because of the thickness of MIP layer with optimal recognising properties is limited in the direction from the membrane surface. It has been shown that AFM and SEM techniques are powerful tools to quantify pore size, surface roughness as well as measure the adhesion force between the MIP and the initial membranes. This provides better understanding of the analysis of composite MIP membranes.

Acknowledgements This work has been supported by grants from the Wellcome Trust (GR 066110/Z/01/Z) and the UK

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