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Molecular imprinted Nylon-6 as a recognition material of amino acids
European Polymer Journal 38 (2002) 521–529 www.elsevier.com/locate/europolj
Molecular imprinted Nylon-6 as a recognition material of amino acids P. Screenivasulu Reddy, Takaomi Kobayashi *, Masanori Abe, Nobuyuki Fujii Department of Chemistry, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka 920-2188, Japan Received 18 September 2000; received in revised form 23 April 2001; accepted 30 July 2001
Abstract Nylon-6 was functionalized by phase inversion molecular imprinting technique to introduce L -glutamine recognition property. For imprinting L -glutamine in the polymer, 20 wt.% of Nylon-6-formic acid solution with 8 wt.% of L glutamine template was used for the phase inversion process in water. The resulted polymer including the template molecule was washed with acetic acid solution to extract the template from the polymer. The substrate binding experiments of the L -glutamine imprinted polymer were examined in aqueous L -glutamine, D -glutamine, L -glutamic acid, and D -glutamic acid solution. The binding of L -glutamine increased with the increase of the amino acid concentration from 5 to 20 lM. The value of equilibrium binding constant for L -glutamine, KE ¼ 4:9 105 M1 , was larger than that for D -glutamine, KE ¼ 1:5 105 M1 . The recognition experiments were extended to membrane filtration and quartzcrystal microbalance response by using the imprinted Nylon-6. Evidence was also presented by FT-IR analysis that the amide–hydrogen-bonding interaction between the imprinted Nylon-6 and template was originated for the amino acid recognition. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Molecularly imprinted polymer; Nylon; Glutamine recognition; Membrane; Sensor
1. Introduction Molecular recognition is becoming an intrinsic part of many biological and chemical processes [1,2]. Molecularly imprinted synthetic polymers have received much attention and extensively studied by many authors during recent years, because of their potential and practical applications for polymeric receptor [3–6] and membrane uses [7–13]. For their molecular imprinting methods, there are two main approaches such as covalent [3] and non-covalent [5] imprinting methods adopted by using cross-linked polymers. In majority of the imprinted cross-linked polymers, non-covalent interactions via hydrogen bonding were based on recognizing the substrate molecules in the polymers. For uses of
*
Corresponding author. Tel.: +81-258-47-9326; fax: +81258-47-9300. E-mail address: [email protected] (T. Kobayashi).
protein having polyamide segments, imprinting process for higher binding capacities was reported [14]. The phenomenon of the imprinted polyamide was extended to both natural and synthetic macromolecules such as polysaccharides and their derivatives, and polymethacrylic acid [15]. On the other hand, synthetic polyamides Nylon have been widely used as affinity supports for Nylon membranes [16] and enzyme immobilization [17]. However, little was known using synthetic Nylon for molecular imprinting. It is worth nothing that Nylon-6 (Scheme 1) has inter- and intra-molecular hydrogen bonding through its amide group (Scheme 2). In our preliminary work, the hydrogen-bonding networks in the polyamides were utilized to molecular imprinting and novel recognition polymer of Nylon-6 was prepared the molecular imprinting Nylon-6 highly recognized amino acids [11]. Selective binding of L -glutamine occurred by the L -glutamine imprinted Nylon. However, the preliminary study only described the binding of the amino acids and another substrates. Therefore, it is
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Scheme 1. Chemical structure of Nylon-6.
Four types of amino acids (Nakarai tesque) such as L -glutamine, D -glutamine, L -glutamic acid, and D -glutamic acid were used as substrates without further purification. Another reagents for experiments were the analytical grade chemicals and were used without further purification. Distilled water passed through ionexchange columns was employed for the preparation of the imprinted polymer and binding experiments of various amino acids to the imprinted polymer.
2.2. Preparation of molecular imprinting Nylon-6 and its characterization
Scheme 2. Hydrogen-bonding networks of Nylon-6.
The molecularly imprinted Nylon-6 was prepared by phase inversion method as reported [11]: Formic acid containing 20 wt.% of Nylon-6 and 8 wt.% of L -glutamine was casted at 50 °C on a glass plate to be about 100 lm thickness. After the solidification of the Nylon-6 in distilled water at 21 °C, the solidified Nylon-6 was well washed with distilled water to remove the solvent and the template from the polymer. The reference polymer without template molecule was also prepared in a similar manner with the imprinted Nylon-6 by using 20 wt.% Nylon-6-formic acid solution. FT-IR spectroscopy (FTIR-8200 spectrometer (Shimadzu, Kyoto, Japan)) was adopted for characterization of the imprinted Nylon-6 before and after template extraction. 2.3. Recognition experiments of amino acids by the glutamine imprinted polymer
Scheme 3. Chemical structure of glutamine.
meaningful to characterize the properties of the molecularly imprinted polymer in details. In the present article, we described properties of the imprinted Nylon-6 as a recognition materials of L -glutamine, which was used for template. To encode the template molecule in the polymer, we applied a phase inversion method [18] and the resultant imprinted polymer was applied for membrane permeation and sensor experiments. In addition, we focused on how the imprinting Nylon-6 was functionalized on the recognition for L -glutamine (Scheme 3) and its racemic analogies. 2. Experimental 2.1. Materials Nylon-6 having molecular weight of 20,000 was produced by Mitsubishi Chemical Co. (Tokyo, Japan).
L-
Binding experiments of amino acids to the L -glutamine imprinted Nylon-6 were performed in 10 ml of amino acid aqueous solution having 5–20 lM concentrations at 30 °C. The change of amino acid concentrations in the aqueous solution was determined by monitoring UV absorbance at 210 nm with UV detector (Tosoh UV8020). Amounts of amino acid bound to the imprinted polymer, ½Sa , were calculated by the following equation: ½Sa ¼ ðCo Ct ÞV =W
ð1Þ
where, Co and Ct were the amino acid concentration (lM), measured at initial and after interval time (hour) for equilibrium. Symbols V and W were the volume of the amino acid solution (10 ml) and the weight of dry polymer (0.015 g) used for the binding experiment, respectively. Selective factor (a) of the imprinted polymer was the relative value of amino acid substrate bound to the imprinted polymer compared with that of L -glutamine template and was calculated by the following formula:
P. Screenivasulu Reddy et al. / European Polymer Journal 38 (2002) 521–529
a ¼ ½Sa s =½Sa sðl-glutamineÞ
ð2Þ
Here, ½Sa s (lmol/g-polymer) was saturation binding amounts of D -glutamine, L - and D -glutamic acid and ½Sa s ðl-glutamineÞ for L -glutamine. 2.4. Permeation experiments of through the imprinted polymers
L -glutamine
solution
A 50 ml capacity of Amicon ultrafiltration cell (UF8050) was used to study the permeation behavior of L or D -glutamine solution. The experimental procedure was followed by previous reports [18]: The L -glutamine imprinted Nylon membrane with 43 mm diameter was mounted on the UF cell and 50 ml of 10 lM amino acid aqueous solution was fed into the cell. Under applied pressure of 1 kg/cm2 the permeated solution through the membrane was collected at different time intervals. The volume flux of the amino acid solution through the polymer was calculated by the following formula [18]: volume flux ðm3 =m2 sÞ ¼ v=ðAtÞ
ð3Þ
where v was the volume of permeate (m3 ), A was the area of membrane ð1:34 103 m2 ) and t was the permeation time (s). 2.5. Amino acids recognition by quartz-crystal microbalance sensor Quartz-crystal microbalance (QCM) sensor response to the amino acid analyte was carried out with Hokutodenko, HQ-304A cell as well as our previously reported paper [19]. The sensor consists of a 25.4 mm diameter disk made from an AT-cut 6 MHz quartzcrystal with gold electrodes (Hokutodenko) on both sides of the crystal. The imprinted Nylon-6 was attached on one side of the quartz-crystal, the other side Au electrode was connected to QCM controller (Hokum dank, HQ-101B) and then sealed by silicon rubber ‘‘O’’ rings between quartz-crystal and QCM holder. The QCM sensor was immersed in various concentrations of L -glutamine aqueous solution (300 ml) at 30 °C and then the frequency change at various times was measured by the QCM controller operated by a microcomputer system (NEC Co., PC9821 Ce2). The details of the QCM sensor was reported in the previous paper [19].
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with those of the L -glutamine analogous such as D glutamine, L -glutamic acid, and D -glutamic acid. Fig. 1 shows the time profiles of the resultant ½Sa for L -glutamine. The binding experiments were performed with various L -glutamine concentrations in the range of 5–20 lM. The values of ½Sa increased with time and became constant at longer time than 12 h. The saturation behavior of the L -glutamine binding indicates that the binding sites of the imprinted polymer were filled with the L -glutamine. Here, the saturation binding amounts of L -glutamine at the 24 h were defined as ½Sa s (lmol/gpolymer). The figure also supported that the saturated binding amounts of ½Sa s were increased, as the amino acid concentration was increased from 5 to 20 lM. For example, the value of ½Sa s was 1.9 and 3.4 lmol/gpolymer for 5 and 20 lM concentration, respectively. While the L -glutamine imprinted polymer could highly bind L -glutamine, the Nylon-6 prepared without molecularly imprinted treatment showed no binding of the amino acid in 20 lM of the L -glutamine concentration (open symbol in the Fig. 1). This means that the resultant Nylon-6 had no binding sites of L -glutamine. Similar binding experiments with L -glutamine were carried out by using the L -glutamine imprinted Nylon-6 for D -glutamine substrate. Fig. 2 presents the time profile of ½Sa observed in different concentrations of D glutamine. The values of ½Sa of D -glutamine increased with time as well as that for L -glutamine. However, the value of ½Sa s for D -glutamine was lower than that measured for L -glutamine. In 20 lM concentration, the ½Sa s was 1.1 lmol/g-polymer for D -glutamine and
3. Results and discussion 3.1. Binding of amino acid to molecularly imprinted Nylon-6 In order to study the recognition characteristics of the L -glutamine imprinted Nylon-6, the binding amounts ½Sa (lmol/g-polymer) of the amino acid were compared
Fig. 1. L -glutamine binding to the L -glutamine imprinted Nylon-6 at various solute concentrations: L -glutamine concentration was shown by symbols of (r) 5 lM, (j) 10 lM, (N) 15 lM, and ( ) 20 lM. Open symbol was for the unimprinted Nylon-6 and the L -glutamine concentration was 20 lM. The binding experiments were carried out at 30 °C.
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Fig. 2. D -glutamine binding to the L -glutamine imprinted Nylon-6 at various solute concentrations: D -glutamine concentration was shown by the same symbols with those shown in Fig. 1. The experiments were similarly performed with the L -glutamine binding.
3.4 lmol/g-polymer for L -glutamine. Here, D -glutamine is an isomer of L -glutamine used as the template in the imprinting process. However, the L -glutamine imprinted polymer bound somewhat D -glutamine. This may be because the imprinted polymer having non-specific binding sites to the D -glutamine molecules. The figure also presents the binding experiments of the unimprinted Nylon-6 in 20 lM D -glutamine solution. However, no D -glutamine binding was similarly observed (open symbol in Fig. 2) with the cases of L -glutamine. Furthermore, recognition experiments of L -glutamic acid and D -glutamic acid were performed at the same concentration range and the value of ½Sa s measured at different concentration was estimated. Fig. 3 shows plots of ½Sa s versus ½Sa 0 , which was an initial concentration of the amino acid solution. It can be seen that the binding curves of L - and D -glutamic acids were almost same as those for D -glutamine. However, it is apparent that the values of ½Sa s for L -glutamine were higher than those of L - and D -glutamic acids and D -glutamine. The results indicate that the L -glutamine imprinted Nylon-6 showed selective recognition on L -glutamine, which was used as a template. It was known that binding constants KE can give a quantitative information about the substrate binding to the polymer [20]. Thus, the amino acid binding into the L -glutamine imprinted Nylon-6 was evaluated by Eq. (4) of Scatchard relationship [20]. ½Sa b ¼ KE ½PSf ¼ KE f½PStotal ½PSb g ½Sa f ¼ KE f½PStotal ½Sa b g
ð4Þ
Fig. 3. Saturation binding curves of L -glutamine ( ), D -glutamine (N), L -glutamic acid (j) and D -glutamic acid (r) for the L -glutamine imprinted Nylon-6. The ½Sa s was the amount (lmol/g-polymer) of amino acid bound to the imprinted polymer. The weight of the imprinted polymer used for the each binding experiments was 15 mg.
Here, ½Sa f and ½Sa b were the concentrations of the substrates free and bound to the polymer sites, respectively. [PS]f was number of free binding sites of polymer, which equals ½PStotal ½PSb , also ½PSb ¼ ½Sa b and [PS]total was the total number of bound sites of polymer. From the binding data, the amounts, ½Sa b of amino acid bound to the known amount of dry polymer was obtained (shown in Fig. 3) by subtracting ½Sa f from the initial amino acid concentration ½Sa 0 ; i.e., ½Sa b ¼ ½Sa 0 ½Sa f . In Eq. (4), [PS]total is the apparent total number of bound sites of substrate amino acid in the polymer. Fig. 4 shows Scatchard plots obtained by binding experiments of amino acids for the L -glutamine imprinted polymer system. Since the plots showed a linear relationship between ½Sa b versus ½Sa b =½Sa f , the negative slope and the intercepts gave each value of KE and ½PStotal . The resulted KE and ½PStotal are summarized in Table 1. Evidently, the values of KE and ½PStotal for L -glutamine were higher than those for the L -glutamine analogous. We estimated also the selectivity factor, a, which is listed in the table. It is evidence for the high selectivity of the polymer that the resultant a for D glutamine was lower than unity for the L -glutamine. Also, the values of a for L - and D -glutamic acids were lower that that of the D -glutamine. Although the unimprinted Nylon had no binding of the glutamine, the L -glutamine imprinted Nylon showed non-specific binding. This difference means that the imprinted process of L -glutamine encoded L -glutamine shape, although the sites slightly showed loose recognition for the D -isomer.
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Fig. 4. Scatchard plots for the binding of L -glutamine and another amino acids by the L -glutamine imprinted Nylon-6. The symbols in the figure were the same as those in Fig. 3 for each amino acid.
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amide-III, amide-IV, and amide-V, respectively [21,22]. We noted the spectra difference of the amide-I and II region in the spectra (b) and (c). As shown by Fig. 6, a variation in the absorption peak height of amide-II stretching was observed before and after the L -glutamine extraction. This is assigned to inter- and intramolecule hydrogen bonding as shown in Scheme 2. The IR absorption band of amide-I at 1642 cm1 became slightly broader than that obtained without the template extraction (b). In addition, the absorption peak of amide-II at 1546 cm1 was slightly shifted to lower wave number side of 1539 cm1 after the template extraction. The peak heights corresponding to amide-II was apparently reduced and the peak widths became slightly broad after the extraction. This indicates that the hydrogen-bonding networks of the Nylon undergo a slight changes before and after the template extraction. Thus, the spectral data obtained here explained that the hydrogen networks between the Nylon chains was the origin to the recognition of the L -glutamine molecules. 3.2. Application of the imprinted Nylon for membrane and QCM sensor
Table 1 Equilibrium constants, maximum binding sites, and template factor (a) of each amino acid substrate KE (M1 )
Substrate L -glutamine D -glutamine L -glutamic D -glutamic
acid acid
5
4:9 10 1:5 105 2:1 105 1:6 105
[PS]total (lmol/g)
a
5.9 2.3 2.1 1.9
1.00 0.39 0.36 0.32
In order to consider the origin of the selectivity of by the imprinted Nylon-6, we previously reported the evidence of infrared (IR) spectra of the polymers. The FT-IR spectra provided an information on the interaction between the imprinted polymer and templates via hydrogen bonding [11]: The IR band of free amide group near 3000 cm1 was monitored. On the other hand, in the present article, the IR bands of amide–carbonyl region at 500–1700 cm1 were measured to evaluate the hydrogen bonding between Nylon-6 polymer chains. Fig. 5 shows the FT-IR spectra of (a) Nylon-6 and L -glutamine imprinted Nylon-6 obtained (b) before and (c) after the template extraction. The spectrum of L -glutamine was also presented as a reference in the figure (d). It was known that the wave number region has a characteristic amide–carbonyl IR absorptions of Nylon. In the IR spectra, the characteristic IR bands of amide-I and amide-II of Nylon-6 were appeared near 1642 and 1546 cm1 , which were assigned to C@O stretching vibration (amide-I) and N–H deformation (amide-II), respectively. There are another absorption peaks near 1265, 695 and 578 cm1 for L -glutamine
We mentioned that the L -glutamine imprinted Nylon-6 exhibits an excellent property of selective absorbent on the L -glutamine molecules. Furthermore, the imprinted Nylon was applied as membrane permeation absorbents and QCM sensors, since these techniques of membrane [13,23,24] and sensor [25,26] were receiving increasing importance in various chemical and biological fields. Before the L -glutamine imprinted Nylon-6 was used in membrane permeation, we confirmed the morphology of the L -glutamine imprinted Nylon-6 by scanning electron micrography (SEM). As previously reported for Nylon-6 membranes prepared with phase inversion process [18], the imprinted polymer had porous structure. Thus, the porous Nylon was successfully applied as membrane materials for low pressure desalination. On the other hand, Fig. 7 depicts SEM pictures of the crosssection and pores of the L -glutamine imprinted Nylon-6. The image implies that the membrane thickness was about 30 lm (a) and the pores (b) were in the range of about 1–2 lm. Therefore, their porous nature of the imprinted Nylon enables glutamine solute to permeate through the membrane. In the experiments, both volume flux and the glutamine concentration were determined at different time intervals after the solution permeation. Fig. 8(a) shows the time course of volume flux of L glutamine, D -glutamine, L -glutamic acid, and D -glutamic acid solution. The values of the flux were 7:8 107 m3 /m2 s at 1 h permeation and were almost same as the value of water. It can be seen that the volume flux was slightly reduced with the increase of the permeation time. At 24 h, the value of L -glutamine solution was
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Fig. 5. FT-IR spectra of the L -glutamine imprinted and unimprinted Nylon-6: (a) Nylon-6 without imprinting and with imprinting (b) before and (c) after the template extraction; (d) was for L -glutamine measured by KBr pellets.
6:7 107 m3 /m2 s. The Fig. 8(a) shows that the reduction tendency was higher in the L -glutamine solution. This may be because of L -glutamine binding to the volumatic space of the template sites by the permeation of the substrate through the membrane. As shown in Fig. 8(b), the binding amounts of the amino acids by the imprinted membrane were increased with the permeation time. After 24 h permeation, the saturation binding was observed. This means that the L -glutamine imprint sites were filled by the amino acid binding. Therefore, in the permeation mode, the L -glutamine imprinted membrane had an effective binding of the L -glutamine solute. Moreover, the amino acid recognition by the L -glutamine imprinted Nylon was examined as QCM sensor application. Up to this point, application of the imprinted polymer for QCM sensor was made using various kinds of imprinted polymers. The sensor design, which was the combination of molecular imprint poly-
mer with QCM, already reported by Mosbach [25], Haupt [26], Kanekiyo [27] and Takeuchi [28]. Most of these QCM sensors contained imprinted polymer layer on Au electrode, where photocrosslinked polymerization was carried out in the presence of template. On the other hand, in our work, phase inversed polymer layer was used and applied for novel type of QCM sensors [19]. In the previous paper of polyacrylonitrile membrane for theophylline recognition, the PAN membrane prepared by the phase inversion imprinting was used. In the present article, the L -glutamine imprinted Nylon-6 membrane was attached on the Au electrode of the QCM. Fig. 9 shows the time course of QCM response measured at various L -glutamine concentrations. The inserted illustration shows the QCM cell with the imprinted membrane, which was immersed in L -glutamine solution and the frequency change (Hz) was monitored with the time. The concentration of the L -glutamine was changed from 0 to 20 lM. From the results, the frequency was
P. Screenivasulu Reddy et al. / European Polymer Journal 38 (2002) 521–529
Fig. 6. FT-IR spectra of Nylon-6 and the imprinted Nylon-6 observed in the region of amide-I and II: (a) Nylon-6 without imprinting and Nylon-6 with imprinting (b) before and (c) after the template extraction.
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Fig. 8. Time profiles of (a) volume flux and (b) ½Sa measured by permeation experiments of L -glutamine ( ), D -glutamine (N), L -glutamic acid (j) and D -glutamic acid (r). Applied pressure was 1 kg/cm2 for the permeation of 10 lM amino acid aqueous solution.
Fig. 7. SEM image of the L -glutamine imprinted Nylon membrane. (a) Cross-section of the imprinted membrane and (b) pores in the membrane. The size of the pictures were shown in the figure.
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mine was confirmed by the imprinted polymer. Evidence for the recognition was supported by FT-IR analysis that the imprinting was performed in the hydrogenbonding networks of the imprinted Nylon-6s. In addition, the L -glutamine recognition was observed in the membrane permeation. QCM response to the L -glutamine was also evaluated by using the imprinted Nylon membrane on the QCM electrode. It was suggested that the L -glutamine imprinted Nylon was applicable for selective absorbent, affinity membrane and sensor.
Acknowledgements
Fig. 9. Frequency change of the L -glutamine imprinted polymer-QCM sensor for L -glutamine and D -glutamine. The concentration of L -glutamine was changed in the range of 5–20 lM. Open symbol was for 20 lM of D -glutamine concentration.
reduced as the time was increased from 0 to 30 min. After about 20–30 min, the value of the frequency was reached to the lowest frequency and showed a constant value. It was observed that the reduction of the frequency depends on the L -glutamine concentration. Note that the high concentration of the L -glutamine caused a large frequency change of the QCM. On the other hand, the frequency reduction for the 20 lM D -glutamine solution was very low relative to that observed in the L glutamines. Therefore, this means that the L -glutamine imprinted Nylon-6 bound the L -glutamine molecules with high affinity. As a result, the frequency was significantly reduced in the L -glutamine solution. From the frequency change measured at various concentrations of L -glutamine, Scatchard analysis was drawn in order to estimate KE . The calculated value for L -glutamine was 5:3 105 M1 and was almost same as obtained in batch experiments for the L -glutamine recognition (Table 1). This means that the sensor experiments would give a new evidence for the application of porous imprinted Nylon membrane in addition to the permeation property.
4. Conclusions Molecular imprinting of L -glutamine was carried out by the phase inversion imprinting of Nylon-6-L -glutamine system. The porous polymer imprinting material was prepared by precipitation of the polymer in the presence of the L -glutamine template in water. After the template extraction from the polymer, the selective L glutamine binding was observed by heterogeneous batch and membrane permeation experiments. In the batch binding experiments, the high recognition of L -gluta-
This work was partially supported by grant-in-aid for scientific research (12650764 and 13027227) from the Ministry of Education, Science, Sports, and Culture, Japan.
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Molecular imprinted Nylon-6 as a recognition material of amino acids P. Screenivasulu Reddy, Takaomi Kobayashi *, Masanori Abe, Nobuyuki Fujii Department of Chemistry, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka 920-2188, Japan Received 18 September 2000; received in revised form 23 April 2001; accepted 30 July 2001
Abstract Nylon-6 was functionalized by phase inversion molecular imprinting technique to introduce L -glutamine recognition property. For imprinting L -glutamine in the polymer, 20 wt.% of Nylon-6-formic acid solution with 8 wt.% of L glutamine template was used for the phase inversion process in water. The resulted polymer including the template molecule was washed with acetic acid solution to extract the template from the polymer. The substrate binding experiments of the L -glutamine imprinted polymer were examined in aqueous L -glutamine, D -glutamine, L -glutamic acid, and D -glutamic acid solution. The binding of L -glutamine increased with the increase of the amino acid concentration from 5 to 20 lM. The value of equilibrium binding constant for L -glutamine, KE ¼ 4:9 105 M1 , was larger than that for D -glutamine, KE ¼ 1:5 105 M1 . The recognition experiments were extended to membrane filtration and quartzcrystal microbalance response by using the imprinted Nylon-6. Evidence was also presented by FT-IR analysis that the amide–hydrogen-bonding interaction between the imprinted Nylon-6 and template was originated for the amino acid recognition. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Molecularly imprinted polymer; Nylon; Glutamine recognition; Membrane; Sensor
1. Introduction Molecular recognition is becoming an intrinsic part of many biological and chemical processes [1,2]. Molecularly imprinted synthetic polymers have received much attention and extensively studied by many authors during recent years, because of their potential and practical applications for polymeric receptor [3–6] and membrane uses [7–13]. For their molecular imprinting methods, there are two main approaches such as covalent [3] and non-covalent [5] imprinting methods adopted by using cross-linked polymers. In majority of the imprinted cross-linked polymers, non-covalent interactions via hydrogen bonding were based on recognizing the substrate molecules in the polymers. For uses of
*
Corresponding author. Tel.: +81-258-47-9326; fax: +81258-47-9300. E-mail address: [email protected] (T. Kobayashi).
protein having polyamide segments, imprinting process for higher binding capacities was reported [14]. The phenomenon of the imprinted polyamide was extended to both natural and synthetic macromolecules such as polysaccharides and their derivatives, and polymethacrylic acid [15]. On the other hand, synthetic polyamides Nylon have been widely used as affinity supports for Nylon membranes [16] and enzyme immobilization [17]. However, little was known using synthetic Nylon for molecular imprinting. It is worth nothing that Nylon-6 (Scheme 1) has inter- and intra-molecular hydrogen bonding through its amide group (Scheme 2). In our preliminary work, the hydrogen-bonding networks in the polyamides were utilized to molecular imprinting and novel recognition polymer of Nylon-6 was prepared the molecular imprinting Nylon-6 highly recognized amino acids [11]. Selective binding of L -glutamine occurred by the L -glutamine imprinted Nylon. However, the preliminary study only described the binding of the amino acids and another substrates. Therefore, it is
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Scheme 1. Chemical structure of Nylon-6.
Four types of amino acids (Nakarai tesque) such as L -glutamine, D -glutamine, L -glutamic acid, and D -glutamic acid were used as substrates without further purification. Another reagents for experiments were the analytical grade chemicals and were used without further purification. Distilled water passed through ionexchange columns was employed for the preparation of the imprinted polymer and binding experiments of various amino acids to the imprinted polymer.
2.2. Preparation of molecular imprinting Nylon-6 and its characterization
Scheme 2. Hydrogen-bonding networks of Nylon-6.
The molecularly imprinted Nylon-6 was prepared by phase inversion method as reported [11]: Formic acid containing 20 wt.% of Nylon-6 and 8 wt.% of L -glutamine was casted at 50 °C on a glass plate to be about 100 lm thickness. After the solidification of the Nylon-6 in distilled water at 21 °C, the solidified Nylon-6 was well washed with distilled water to remove the solvent and the template from the polymer. The reference polymer without template molecule was also prepared in a similar manner with the imprinted Nylon-6 by using 20 wt.% Nylon-6-formic acid solution. FT-IR spectroscopy (FTIR-8200 spectrometer (Shimadzu, Kyoto, Japan)) was adopted for characterization of the imprinted Nylon-6 before and after template extraction. 2.3. Recognition experiments of amino acids by the glutamine imprinted polymer
Scheme 3. Chemical structure of glutamine.
meaningful to characterize the properties of the molecularly imprinted polymer in details. In the present article, we described properties of the imprinted Nylon-6 as a recognition materials of L -glutamine, which was used for template. To encode the template molecule in the polymer, we applied a phase inversion method [18] and the resultant imprinted polymer was applied for membrane permeation and sensor experiments. In addition, we focused on how the imprinting Nylon-6 was functionalized on the recognition for L -glutamine (Scheme 3) and its racemic analogies. 2. Experimental 2.1. Materials Nylon-6 having molecular weight of 20,000 was produced by Mitsubishi Chemical Co. (Tokyo, Japan).
L-
Binding experiments of amino acids to the L -glutamine imprinted Nylon-6 were performed in 10 ml of amino acid aqueous solution having 5–20 lM concentrations at 30 °C. The change of amino acid concentrations in the aqueous solution was determined by monitoring UV absorbance at 210 nm with UV detector (Tosoh UV8020). Amounts of amino acid bound to the imprinted polymer, ½Sa , were calculated by the following equation: ½Sa ¼ ðCo Ct ÞV =W
ð1Þ
where, Co and Ct were the amino acid concentration (lM), measured at initial and after interval time (hour) for equilibrium. Symbols V and W were the volume of the amino acid solution (10 ml) and the weight of dry polymer (0.015 g) used for the binding experiment, respectively. Selective factor (a) of the imprinted polymer was the relative value of amino acid substrate bound to the imprinted polymer compared with that of L -glutamine template and was calculated by the following formula:
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a ¼ ½Sa s =½Sa sðl-glutamineÞ
ð2Þ
Here, ½Sa s (lmol/g-polymer) was saturation binding amounts of D -glutamine, L - and D -glutamic acid and ½Sa s ðl-glutamineÞ for L -glutamine. 2.4. Permeation experiments of through the imprinted polymers
L -glutamine
solution
A 50 ml capacity of Amicon ultrafiltration cell (UF8050) was used to study the permeation behavior of L or D -glutamine solution. The experimental procedure was followed by previous reports [18]: The L -glutamine imprinted Nylon membrane with 43 mm diameter was mounted on the UF cell and 50 ml of 10 lM amino acid aqueous solution was fed into the cell. Under applied pressure of 1 kg/cm2 the permeated solution through the membrane was collected at different time intervals. The volume flux of the amino acid solution through the polymer was calculated by the following formula [18]: volume flux ðm3 =m2 sÞ ¼ v=ðAtÞ
ð3Þ
where v was the volume of permeate (m3 ), A was the area of membrane ð1:34 103 m2 ) and t was the permeation time (s). 2.5. Amino acids recognition by quartz-crystal microbalance sensor Quartz-crystal microbalance (QCM) sensor response to the amino acid analyte was carried out with Hokutodenko, HQ-304A cell as well as our previously reported paper [19]. The sensor consists of a 25.4 mm diameter disk made from an AT-cut 6 MHz quartzcrystal with gold electrodes (Hokutodenko) on both sides of the crystal. The imprinted Nylon-6 was attached on one side of the quartz-crystal, the other side Au electrode was connected to QCM controller (Hokum dank, HQ-101B) and then sealed by silicon rubber ‘‘O’’ rings between quartz-crystal and QCM holder. The QCM sensor was immersed in various concentrations of L -glutamine aqueous solution (300 ml) at 30 °C and then the frequency change at various times was measured by the QCM controller operated by a microcomputer system (NEC Co., PC9821 Ce2). The details of the QCM sensor was reported in the previous paper [19].
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with those of the L -glutamine analogous such as D glutamine, L -glutamic acid, and D -glutamic acid. Fig. 1 shows the time profiles of the resultant ½Sa for L -glutamine. The binding experiments were performed with various L -glutamine concentrations in the range of 5–20 lM. The values of ½Sa increased with time and became constant at longer time than 12 h. The saturation behavior of the L -glutamine binding indicates that the binding sites of the imprinted polymer were filled with the L -glutamine. Here, the saturation binding amounts of L -glutamine at the 24 h were defined as ½Sa s (lmol/gpolymer). The figure also supported that the saturated binding amounts of ½Sa s were increased, as the amino acid concentration was increased from 5 to 20 lM. For example, the value of ½Sa s was 1.9 and 3.4 lmol/gpolymer for 5 and 20 lM concentration, respectively. While the L -glutamine imprinted polymer could highly bind L -glutamine, the Nylon-6 prepared without molecularly imprinted treatment showed no binding of the amino acid in 20 lM of the L -glutamine concentration (open symbol in the Fig. 1). This means that the resultant Nylon-6 had no binding sites of L -glutamine. Similar binding experiments with L -glutamine were carried out by using the L -glutamine imprinted Nylon-6 for D -glutamine substrate. Fig. 2 presents the time profile of ½Sa observed in different concentrations of D glutamine. The values of ½Sa of D -glutamine increased with time as well as that for L -glutamine. However, the value of ½Sa s for D -glutamine was lower than that measured for L -glutamine. In 20 lM concentration, the ½Sa s was 1.1 lmol/g-polymer for D -glutamine and
3. Results and discussion 3.1. Binding of amino acid to molecularly imprinted Nylon-6 In order to study the recognition characteristics of the L -glutamine imprinted Nylon-6, the binding amounts ½Sa (lmol/g-polymer) of the amino acid were compared
Fig. 1. L -glutamine binding to the L -glutamine imprinted Nylon-6 at various solute concentrations: L -glutamine concentration was shown by symbols of (r) 5 lM, (j) 10 lM, (N) 15 lM, and ( ) 20 lM. Open symbol was for the unimprinted Nylon-6 and the L -glutamine concentration was 20 lM. The binding experiments were carried out at 30 °C.
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Fig. 2. D -glutamine binding to the L -glutamine imprinted Nylon-6 at various solute concentrations: D -glutamine concentration was shown by the same symbols with those shown in Fig. 1. The experiments were similarly performed with the L -glutamine binding.
3.4 lmol/g-polymer for L -glutamine. Here, D -glutamine is an isomer of L -glutamine used as the template in the imprinting process. However, the L -glutamine imprinted polymer bound somewhat D -glutamine. This may be because the imprinted polymer having non-specific binding sites to the D -glutamine molecules. The figure also presents the binding experiments of the unimprinted Nylon-6 in 20 lM D -glutamine solution. However, no D -glutamine binding was similarly observed (open symbol in Fig. 2) with the cases of L -glutamine. Furthermore, recognition experiments of L -glutamic acid and D -glutamic acid were performed at the same concentration range and the value of ½Sa s measured at different concentration was estimated. Fig. 3 shows plots of ½Sa s versus ½Sa 0 , which was an initial concentration of the amino acid solution. It can be seen that the binding curves of L - and D -glutamic acids were almost same as those for D -glutamine. However, it is apparent that the values of ½Sa s for L -glutamine were higher than those of L - and D -glutamic acids and D -glutamine. The results indicate that the L -glutamine imprinted Nylon-6 showed selective recognition on L -glutamine, which was used as a template. It was known that binding constants KE can give a quantitative information about the substrate binding to the polymer [20]. Thus, the amino acid binding into the L -glutamine imprinted Nylon-6 was evaluated by Eq. (4) of Scatchard relationship [20]. ½Sa b ¼ KE ½PSf ¼ KE f½PStotal ½PSb g ½Sa f ¼ KE f½PStotal ½Sa b g
ð4Þ
Fig. 3. Saturation binding curves of L -glutamine ( ), D -glutamine (N), L -glutamic acid (j) and D -glutamic acid (r) for the L -glutamine imprinted Nylon-6. The ½Sa s was the amount (lmol/g-polymer) of amino acid bound to the imprinted polymer. The weight of the imprinted polymer used for the each binding experiments was 15 mg.
Here, ½Sa f and ½Sa b were the concentrations of the substrates free and bound to the polymer sites, respectively. [PS]f was number of free binding sites of polymer, which equals ½PStotal ½PSb , also ½PSb ¼ ½Sa b and [PS]total was the total number of bound sites of polymer. From the binding data, the amounts, ½Sa b of amino acid bound to the known amount of dry polymer was obtained (shown in Fig. 3) by subtracting ½Sa f from the initial amino acid concentration ½Sa 0 ; i.e., ½Sa b ¼ ½Sa 0 ½Sa f . In Eq. (4), [PS]total is the apparent total number of bound sites of substrate amino acid in the polymer. Fig. 4 shows Scatchard plots obtained by binding experiments of amino acids for the L -glutamine imprinted polymer system. Since the plots showed a linear relationship between ½Sa b versus ½Sa b =½Sa f , the negative slope and the intercepts gave each value of KE and ½PStotal . The resulted KE and ½PStotal are summarized in Table 1. Evidently, the values of KE and ½PStotal for L -glutamine were higher than those for the L -glutamine analogous. We estimated also the selectivity factor, a, which is listed in the table. It is evidence for the high selectivity of the polymer that the resultant a for D glutamine was lower than unity for the L -glutamine. Also, the values of a for L - and D -glutamic acids were lower that that of the D -glutamine. Although the unimprinted Nylon had no binding of the glutamine, the L -glutamine imprinted Nylon showed non-specific binding. This difference means that the imprinted process of L -glutamine encoded L -glutamine shape, although the sites slightly showed loose recognition for the D -isomer.
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Fig. 4. Scatchard plots for the binding of L -glutamine and another amino acids by the L -glutamine imprinted Nylon-6. The symbols in the figure were the same as those in Fig. 3 for each amino acid.
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amide-III, amide-IV, and amide-V, respectively [21,22]. We noted the spectra difference of the amide-I and II region in the spectra (b) and (c). As shown by Fig. 6, a variation in the absorption peak height of amide-II stretching was observed before and after the L -glutamine extraction. This is assigned to inter- and intramolecule hydrogen bonding as shown in Scheme 2. The IR absorption band of amide-I at 1642 cm1 became slightly broader than that obtained without the template extraction (b). In addition, the absorption peak of amide-II at 1546 cm1 was slightly shifted to lower wave number side of 1539 cm1 after the template extraction. The peak heights corresponding to amide-II was apparently reduced and the peak widths became slightly broad after the extraction. This indicates that the hydrogen-bonding networks of the Nylon undergo a slight changes before and after the template extraction. Thus, the spectral data obtained here explained that the hydrogen networks between the Nylon chains was the origin to the recognition of the L -glutamine molecules. 3.2. Application of the imprinted Nylon for membrane and QCM sensor
Table 1 Equilibrium constants, maximum binding sites, and template factor (a) of each amino acid substrate KE (M1 )
Substrate L -glutamine D -glutamine L -glutamic D -glutamic
acid acid
5
4:9 10 1:5 105 2:1 105 1:6 105
[PS]total (lmol/g)
a
5.9 2.3 2.1 1.9
1.00 0.39 0.36 0.32
In order to consider the origin of the selectivity of by the imprinted Nylon-6, we previously reported the evidence of infrared (IR) spectra of the polymers. The FT-IR spectra provided an information on the interaction between the imprinted polymer and templates via hydrogen bonding [11]: The IR band of free amide group near 3000 cm1 was monitored. On the other hand, in the present article, the IR bands of amide–carbonyl region at 500–1700 cm1 were measured to evaluate the hydrogen bonding between Nylon-6 polymer chains. Fig. 5 shows the FT-IR spectra of (a) Nylon-6 and L -glutamine imprinted Nylon-6 obtained (b) before and (c) after the template extraction. The spectrum of L -glutamine was also presented as a reference in the figure (d). It was known that the wave number region has a characteristic amide–carbonyl IR absorptions of Nylon. In the IR spectra, the characteristic IR bands of amide-I and amide-II of Nylon-6 were appeared near 1642 and 1546 cm1 , which were assigned to C@O stretching vibration (amide-I) and N–H deformation (amide-II), respectively. There are another absorption peaks near 1265, 695 and 578 cm1 for L -glutamine
We mentioned that the L -glutamine imprinted Nylon-6 exhibits an excellent property of selective absorbent on the L -glutamine molecules. Furthermore, the imprinted Nylon was applied as membrane permeation absorbents and QCM sensors, since these techniques of membrane [13,23,24] and sensor [25,26] were receiving increasing importance in various chemical and biological fields. Before the L -glutamine imprinted Nylon-6 was used in membrane permeation, we confirmed the morphology of the L -glutamine imprinted Nylon-6 by scanning electron micrography (SEM). As previously reported for Nylon-6 membranes prepared with phase inversion process [18], the imprinted polymer had porous structure. Thus, the porous Nylon was successfully applied as membrane materials for low pressure desalination. On the other hand, Fig. 7 depicts SEM pictures of the crosssection and pores of the L -glutamine imprinted Nylon-6. The image implies that the membrane thickness was about 30 lm (a) and the pores (b) were in the range of about 1–2 lm. Therefore, their porous nature of the imprinted Nylon enables glutamine solute to permeate through the membrane. In the experiments, both volume flux and the glutamine concentration were determined at different time intervals after the solution permeation. Fig. 8(a) shows the time course of volume flux of L glutamine, D -glutamine, L -glutamic acid, and D -glutamic acid solution. The values of the flux were 7:8 107 m3 /m2 s at 1 h permeation and were almost same as the value of water. It can be seen that the volume flux was slightly reduced with the increase of the permeation time. At 24 h, the value of L -glutamine solution was
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Fig. 5. FT-IR spectra of the L -glutamine imprinted and unimprinted Nylon-6: (a) Nylon-6 without imprinting and with imprinting (b) before and (c) after the template extraction; (d) was for L -glutamine measured by KBr pellets.
6:7 107 m3 /m2 s. The Fig. 8(a) shows that the reduction tendency was higher in the L -glutamine solution. This may be because of L -glutamine binding to the volumatic space of the template sites by the permeation of the substrate through the membrane. As shown in Fig. 8(b), the binding amounts of the amino acids by the imprinted membrane were increased with the permeation time. After 24 h permeation, the saturation binding was observed. This means that the L -glutamine imprint sites were filled by the amino acid binding. Therefore, in the permeation mode, the L -glutamine imprinted membrane had an effective binding of the L -glutamine solute. Moreover, the amino acid recognition by the L -glutamine imprinted Nylon was examined as QCM sensor application. Up to this point, application of the imprinted polymer for QCM sensor was made using various kinds of imprinted polymers. The sensor design, which was the combination of molecular imprint poly-
mer with QCM, already reported by Mosbach [25], Haupt [26], Kanekiyo [27] and Takeuchi [28]. Most of these QCM sensors contained imprinted polymer layer on Au electrode, where photocrosslinked polymerization was carried out in the presence of template. On the other hand, in our work, phase inversed polymer layer was used and applied for novel type of QCM sensors [19]. In the previous paper of polyacrylonitrile membrane for theophylline recognition, the PAN membrane prepared by the phase inversion imprinting was used. In the present article, the L -glutamine imprinted Nylon-6 membrane was attached on the Au electrode of the QCM. Fig. 9 shows the time course of QCM response measured at various L -glutamine concentrations. The inserted illustration shows the QCM cell with the imprinted membrane, which was immersed in L -glutamine solution and the frequency change (Hz) was monitored with the time. The concentration of the L -glutamine was changed from 0 to 20 lM. From the results, the frequency was
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Fig. 6. FT-IR spectra of Nylon-6 and the imprinted Nylon-6 observed in the region of amide-I and II: (a) Nylon-6 without imprinting and Nylon-6 with imprinting (b) before and (c) after the template extraction.
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Fig. 8. Time profiles of (a) volume flux and (b) ½Sa measured by permeation experiments of L -glutamine ( ), D -glutamine (N), L -glutamic acid (j) and D -glutamic acid (r). Applied pressure was 1 kg/cm2 for the permeation of 10 lM amino acid aqueous solution.
Fig. 7. SEM image of the L -glutamine imprinted Nylon membrane. (a) Cross-section of the imprinted membrane and (b) pores in the membrane. The size of the pictures were shown in the figure.
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mine was confirmed by the imprinted polymer. Evidence for the recognition was supported by FT-IR analysis that the imprinting was performed in the hydrogenbonding networks of the imprinted Nylon-6s. In addition, the L -glutamine recognition was observed in the membrane permeation. QCM response to the L -glutamine was also evaluated by using the imprinted Nylon membrane on the QCM electrode. It was suggested that the L -glutamine imprinted Nylon was applicable for selective absorbent, affinity membrane and sensor.
Acknowledgements
Fig. 9. Frequency change of the L -glutamine imprinted polymer-QCM sensor for L -glutamine and D -glutamine. The concentration of L -glutamine was changed in the range of 5–20 lM. Open symbol was for 20 lM of D -glutamine concentration.
reduced as the time was increased from 0 to 30 min. After about 20–30 min, the value of the frequency was reached to the lowest frequency and showed a constant value. It was observed that the reduction of the frequency depends on the L -glutamine concentration. Note that the high concentration of the L -glutamine caused a large frequency change of the QCM. On the other hand, the frequency reduction for the 20 lM D -glutamine solution was very low relative to that observed in the L glutamines. Therefore, this means that the L -glutamine imprinted Nylon-6 bound the L -glutamine molecules with high affinity. As a result, the frequency was significantly reduced in the L -glutamine solution. From the frequency change measured at various concentrations of L -glutamine, Scatchard analysis was drawn in order to estimate KE . The calculated value for L -glutamine was 5:3 105 M1 and was almost same as obtained in batch experiments for the L -glutamine recognition (Table 1). This means that the sensor experiments would give a new evidence for the application of porous imprinted Nylon membrane in addition to the permeation property.
4. Conclusions Molecular imprinting of L -glutamine was carried out by the phase inversion imprinting of Nylon-6-L -glutamine system. The porous polymer imprinting material was prepared by precipitation of the polymer in the presence of the L -glutamine template in water. After the template extraction from the polymer, the selective L glutamine binding was observed by heterogeneous batch and membrane permeation experiments. In the batch binding experiments, the high recognition of L -gluta-
This work was partially supported by grant-in-aid for scientific research (12650764 and 13027227) from the Ministry of Education, Science, Sports, and Culture, Japan.
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