ARTICLE pubs.acs.org/ac
Enhanced Lysozyme Imprinting Over Nanoparticles Functionalized with Carboxyl Groups for Noncovalent Template Sorption Guoqi Fu,* Hongyan He, Zhihua Chai, Huachang Chen, Juan Kong, Yan Wang, and Yizhe Jiang Key Laboratory of Functional Polymer Materials of Ministry of Education, Institute of Polymer Chemistry, Nankai University, Tianjin 300071, People’s Republic of China
bS Supporting Information ABSTRACT: Surface molecular imprinting, in particular over nanosized support materials, is very suitable for a template of bulky structure like protein. Inspired by the surface template immobilization method reported previously, we herein demonstrate an alternative strategy for enhancing specific recognition of core-shell protein-imprinted nanoparticles through prefunctionalizing the cores with noncovalent template sorption groups. For proof of this concept, silica nanoparticles chosen as the core materials were modified consecutively with 3-aminopropyltrimethoxysilane and maleic anhydride to introduce polymerizable double bonds and terminal carboxyl groups, hence capable of physically adsorbing the print protein. With lysozyme as a template, thin protein-imprinted shells were fabricated according to our newly developed approach for surface protein imprinting over nanoparticles. The rebinding experiments confirmed that the introduction of the carboxyl groups could remarkably improve the imprinting effect in relation to a significantly increased imprinting factor and specific rebinding capacity. Moreover, in contrast to the harsh template removal conditions required for the covalent template coupling approach, the template removal during the imprinted particle synthesis as well as desorption after rebinding could be mildly achieved via washing with salt solution.
M
olecular imprinting is an inexpensive method for the synthesis of tailor-made recognition materials by copolymerizing suitable functional monomers in the presence of desired template molecules. In comparison with natural recognition materials like antibodies, molecularly imprinted polymers (MIPs) offer advantages like stability, specific recognition, and ease of mass preparation, and thus have found applications in wide areas including separation, sensors, and catalysis.1-3 Apart from the relatively established imprinting of low-molecular-weight compounds, these years the MIPs against proteins and other biologically relevant targets have attracted increasing research interest.4-7 This can be attributed to their potential as substitutes for the expensive and labile antibodies, which are now widely used in the areas such as biosensors, bioseparation, and medical diagnostics. As for protein imprinting, the major problem lies in their restricted mass transfer across the cross-linked polymer matrix due to the large molecular sizes, which restricts the ease of template removal as well as rebinding. Other problems include complex and flexible structures of proteins and significantly reduced noncovalent template-monomer interactions in aqueous media where proteins prefer to exist, all of which are disadvantageous to the creation of high-quality imprinting sites for selective template recognition.8-10 Considering the mass transfer difficulty, a variety of approaches have been developed, for example, surface imprinting,9,11 epitope imprinting with a fragment of the original macromolecular target as a template,12,13 and the use of moderately cross-linked hydrogels.14,15 Among these, surface imprinting with the imprinted sites situated at r 2011 American Chemical Society
or close to the surface of MIPs, enabling easy access to the target protein molecules, is accepted to be the most promising for protein template. Surface imprinting of proteins has been extensively studied over silica microparticles and other support materials based on a variety of ways for the formation of surface coatings, e.g., radicalinduced graft polymerization,16 sol-gel transformation,9 self-polymerization of 3-aminophenylboronic acid or dopamine,17,18 fixation of self-assembled monolayers (SAMs),19,20 formation of polymer membranes via phase inversion without polymerization,21 and using polymer brush via solvent-assisted polymer grafting.22 On the other hand, nanostructured MIPs have a small dimension with extremely high surface-to-volume ratio so that most of template molecules are situated at the surface or in the proximity of the material’s surface. Obviously, nanosized MIPs are expected to improve accessibility of the recognition sites generated and, hence, the binding capacity and binding kinetics.23,24 Recently, different nanotechniques have been explored for the imprinting of protein molecules.10,18,25-31 Among those, the strategies based on radical-initiated polymerization28-31 may be more versatile, since various (methyl)acrylic functional monomers can be chosen according to the properties of the protein to be printed. Relatively fewer approaches have been developed thus far to improve the recognition selectivity of protein-imprinted polymers. Received: November 13, 2010 Accepted: January 7, 2011 Published: January 25, 2011 1431
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Analytical Chemistry The specificity can be increased to varied extent by combined use of the functional monomers with different groups and optimizing the composition and ratio32 or by using monomers with moieties already known to interact relatively strong with the target protein, e.g., taking advantage of enzyme-inhibitor and metal coordination interactions.29 Shiomi et al.9 pioneered a strategy based on covalent template immobilization combined with surface imprinting. Target protein molecules were covalently attached to the surface of aldehyde group modified silica microspheres through reversible imine linkage, and then imprinted layers were coated thereon using silanes via a sol-gel process, followed by the template removal step which involved destroying the formed covalent bonds by washing with oxalic acid solution for more than 10 h. This method has been adapted to various support materials and different surface coating process by several groups.10,13,17,27,33 As-prepared core-shell MIPs often show increased template rebinding selectivity and capacity compared to those fabricated with free template. This can be attributed to the higher template density attached on the support surface and the additional covalent interaction forces exerted on the template while forming the imprinting sites. However, this method has a serious disadvantage with respect to the rather harsh conditions required for template removal which would affect the imprint stability, unless the template is stripped following sacrificing the supporting materials.10,13 This may be responsible for the fact that, until now, there has been no report on the repeated use of this type of imprinted material. Inspired by such a template immobilization strategy, we propose to introduce a functional group other than an aldehyde onto the support surface which can physically adsorb the template protein from the prepolymerization solution. During subsequent formation of surface imprinting layers, this group would also contribute to the construction of the imprinting sites. Therefore, in a similar way, the imprinting effect of the resulting MIPs would also be enhanced. More importantly, the template removal or elution would become facilitated and the repeated use of the MIPs hence more feasible. In a recent publication,34 we demonstrated a facile approach for surface protein imprinting over vinyl-modified silica nanoparticles via surface graft copolymerization using low monomer concentration. To test the above hypothesis, herein we introduced an additional carboxyl group onto the nanosupport surface, which can interact with lysozyme (Lys), a model protein chosen as the template, via electrostatic forces. The structure and morphology of the resulting imprinted particles were characterized by transmission electron microscopy (TEM) and thermogravimetric analysis (TGA). The protein recognition properties were examined by single-protein or competitive batch rebinding experiments and rebinding kinetics study. The regeneration of the imprinted particles after rebinding and recognition reproducibility was also studied.
’ EXPERIMENTAL SECTION Reagents. Maleic anhydride of analytical grade were purchased from Tianjin Chemical Reagents Company. 3-Aminopropyltrimethoxysilane (APTMS) was purchased from Chemical Factory of Wuhan University (Wuhan, China). All other chemicals were obtained and treated as in our previous work.34 Synthesis of Functionalized Silica Nanoparticles. Monodisperse silica nanoparticles were synthesized according to the reported St€ober method.35 The silica particles were then modified successively with APTMS and maleic anhydride to introduce polymerizable double bonds and terminal carboxyl groups.
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Briefly, 2 g of the silica nanparticles was dispersed in 50 mL of anhydrous toluene containing 2 mL of APTMS, and the mixture was allowed to react at 70 °C for 8 h under dry nitrogen. The resultant APTMS-functionalized silica particles (denoted as silica-NH2) were separated by centrifugation and washed repeatedly with toluene and acetone. The silica-NH2 particles were redispersed in 50 mL of anhydrous N,N0 -dimethylformamide containing maleic anhydride (2 g) and pyridine (5 mL), and the dispersion was heated to and kept at 50 °C under nitrogen atmosphere for 3 h. The maleic acid modified particles (denoted as silica-COOH) were gathered by centrifugation, washed extensively with ethanol and water, and finally freeze-dried for further use. Synthesis of Protein-Imprinted Nanoparticles. With Lys as a template and the silica-COOH particles as cores, the surface Lys-imprinted nanoparticles were synthesized according the protocol described in ref 34 with minor modification, where 3-methacryloxypropyl trimethoxysilane (MPS) modified silica nanoparticles (denoted as silica-MPS) were employed as the supports. Tris buffer (pH 7.0, 0.01 M) was used instead of the ethanol-containing phosphate buffer (pH 7.0, 0.02 M). The control nonimprinted polymers (NIPs) were prepared in the same way but without addition of the template. Protein Adsorption Experiments. The batch rebinding tests, competitive batch rebinding tests, and rebinding kinetics study were performed in the same way as described in ref 34, except that Tris buffer (pH 7.0, 0.01 M) was utilized. The amount of protein adsorbed by the particles at the end of each batch incubation run was calculated from the following formula: q ¼ ðCi -Cf ÞV =m where q (mg/g) is the mass of protein adsorbed by unit mass of dry particles, Ci (mg/mL) and Cf (mg/mL) are the protein concentrations of the initial and final solutions, respectively, V (mL) is the total volume of the adsorption mixture, and m is the mass of the particles used. All the tests were conducted in triplicate. Characterization. The morphologies and structures of the nanoparticles modified differently were examined by a TEM (JEM-2100, JEOL). The ζ-potentials of the particles dispersed in deionized water were measured with a ζ-potential analyzer (Zetasizer Nano ZS, Malvern). TGA was carried out using a thermogravimetric analyzer (TG 209, Netzsch) under nitrogen atmosphere with a heating rate of 10 °C/min up to 900 °C.
’ RESULTS AND DISCUSSION Synthesis and Characterization of Lys-Imprinted Silica Nanoparticles. The surface protein imprinting strategy based
on preimmobilization of protein template on support materials, first reported by Shiomi et al.,9 has been proved to be effective often with improved template selection and affinity of the resulting MIPs compared to the traditional free-template methods. However, the template removal or desorption process involves incubating the MIPs in harsh acidic or basic conditions for up to 10 h. This drawback greatly restricts the applicability of this approach, particularly when the MIPs have to be used repeatedly or the template needs to be recovered. Motivated by such a concept of covalent template immobilization, we hypothesize that the introduction of the functional groups capable of capturing the template noncovalently, rather than covalently, onto the support surface would overcome the disadvantage, while maintaining the advantages to some degree. Moreover, we have recently reported a new surface protein imprinting method over nanoparticles, and the results suggested that the 1432
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Figure 1. Schematic illustration of silica nanoparticle surface modified differently: (a) with 3-methacryloxypropyl trimethoxysilane; (b) first with 3-aminopropyltrimethoxysilane and subsequently with maleic anhydride.
imprinting effect may be further enhanced through elaborate surface chemistry design of the nanoparticle supports.34 Therefore, the above hypothesis was herein incorporated into our protein imprinting approach. In our previous work,34 Lys was imprinted over silica-MPS particles (see Figure 1a) via radical-induced surface graft copolymerization of acrylamide (AAm) combined with other two oppositely charged monomers (equimolar) and a cross-linker in dilute aqueous solution. Besides AAm, the functional and crosslinking monomers used were negatively charged methacrylic acid (MAA), positively charged 2-(dimethylamino)ethyl methacrylate (DMAEMA), and N,N0 -methylenebisacrylamide (MBA), respectively. The monomer composition was the same as the prepolymerization recipes for the synthesis of Lys-imprinted hydrogels reported by other research groups,15,32 but adapted with a much reduced concentration (0.4 wt %) to avoid the possible gelation of the reaction dispersion. Nevertheless, the imprinted polymer shell layers can be formed over the silica-MPS particles. The reactive vinyl groups on the support surface may play an important role for the formation of the polymer shells through the continual capture of the monomers and newly formed oligomers in the solution during polymerization. In this work, we adopted a different surface functionalization protocol. The silica particles were first reacted with APTMS and then with maleic anhydrous to obtain silica-NH2 and silicaCOOH (see Figure 1b), respectively. In comparison with the silica-MPS particles, apart from polymerizable double bonds, the silica-COOH particles bear terminal carboxylic groups which can physically interact with protein template. As seen from Table S-1 in the Supporting Information, the particles carrying different groups exhibited noticeable variation of ζ-potential and Lys sorption, hence confirming the successful surface modification. When calculated from about 50 particles in the TEM images (see Figure 2c), the average particle size of the silica-COOH is 226 nm, about 10% smaller than that of the silica-MPS (248 nm). With the silica-COOH particles as support materials, surface imprinting of Lys was achieved in aqueous monomer solution in the same way as with the silicaMPS in ref 34. Similar to the previous observations, the reaction mixture showed no noticeable gelation after the polymerization, and hence the resulting MIP particles as well as the NIP particles could be readily gathered by centrifugation. Also, both the MIP and NIP particles could be dispersed separately in water or ethanol and inherited the monodispersibility of the original
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silica-COOH particles (see TEM images in Figure 2). Different from the results in ref 34, however, now both the MIP and NIP particles showed a distinct core-shell structure with shell thickness of ∼3 nm, as can be seen from the insets of Figure 2. Though very thin in the dry state, the shell layers of the MIP particles in aqueous media might be swollen to a dimension large enough for accommodating Lys molecules (4.5 nm 3 nm 3 nm). On the basis of the TGA results (see Figure S-1 in the Supporting Information), the MIP particles contained ∼6.7 wt % of grafted polymers, nearly twice that of those which were prepared with silica-MPS as cores.34 This may be explained by assuming that the silica-COOH particles are more hydrophilic than the silica-MPS particles and hence facilitate more the graft copolymerization of hydrophilic monomers like AAm, MAA, and DMAEMA. Quite recently, Gai et al.30 reported surface imprinting of Lys over magnetic silica nanoparticles based on surface-initiated atom transfer radical polymerization (ATRP) of AAm and Nisopropylacrylamide. The resulting imprinted particles showed unagglomerated as expected since the controllable ATRP was confined only to the support surface. However, this technique is limited to the use of some functional monomers like AAm, which have little influence on the catalyst,36 and thus lacks versatility for protein imprinting. Jing et al.31 also demonstrated surface Lys imprinting over MPS-modified magnetic silica nanoparticles with a method very similar to ours in ref 34 but using a much higher monomer concentration (∼7 wt %). The obtained imprinted particles were seriously conglomerated. With such a high monomer concentration, the polymerization mixture should become a gelled monolith after initiation, as we observed experimentally. Unfortunately, the authors did not address this important issue in the article. Rebinding Capacity and Kinetics. The imprinting effect of a MIP is often evaluated by the imprinting factor (IF) and specific adsorption capacity, with the former defined as the ratio of rebinding capacity of the MIP with respect to that of the NIP, and the latter defined as the corresponding difference. Generally, the imprinting factor and specific adsorption capacity reflect the rebinding specificity and imprint amount created of a MIP, respectively. In characterizing the adsorption behaviors of the MIP and NIP nanoparticles, they were subjected to rebinding equilibrium and kinetics studies with a batch binding approach. Since NaCl added in the binding solution may suppress the nonspecific adsorption to the MIPs, we first examined its effect on Lys rebinding to the MIP and NIP particles (Figure S-2 in the Supporting Information). When the solution contained 40 mM NaCl, the MIP particles exhibited both a relatively high imprinting factor and a relatively large specific adsorption capacity. Also, the ion strength of the solution approximates that of the phosphate buffer employed in the rebinding experiments with the silica-MPS-based imprinted particles described in ref 34. Thus, all the following rebinding tests were conducted using the Tris buffer (pH 7.0, 0.01 M) containing 40 mM NaCl for the convenience of comparison. Batch rebinding tests were performed at different initial concentrations of Lys, ranging from 0.1 to 1.0 mg/L. Figure 3 shows the rebinding capacities for Lys at different equilibrium concentrations, i.e., adsorption isotherms. Within the concentration range covered, the MIP particles exhibited much higher binding amounts than the control NIP particles, suggesting that the molecular recognition sites were generated on the surface of the MIP particles by the Lys template involved in the polymerization process. The Langmuir model 1433
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Figure 2. TEM images of (a) Lys-imprinted particles, (b) nonimprinted particles, and (c) silica particles modified with 3-aminopropyltrimethoxysilane and subsequently with maleic anhydride. The insets are the high-resolution pictures with a bar of 20 nm.
Figure 3. Adsorption isotherms of Lys on the imprinted particles and nonimprinted particles. Adsorption conditions: V = 1.5 mL, m = 7.0 mg, Ci = 0.1-1.0 mg/mL, CNaCl = 40 mM, time 1 h, temperature 25 °C, Tris buffer (10 mM, pH 7.0).
was employed to represent the rebinding isotherms, which is expressed as qe ¼ qm Ce =ðKd þCe Þ where Ce (mg/mL) is the equilibrium concentration of Lys in bulk solution, qe (mg/g) is the amount of adsorbed Lys on per gram of the MIP or NIP particles at the equilibrium concentration, qm (mg/g) is the saturation capacity, and Kd (mg/mL) is the dissociate constant. Nonlinear fitting of these data to the equation yielded a good fit for the MIP and NIP particles (with the regression coefficient higher than 0.97), and the involved parameters were determined. Listed in Table 1 are the obtained parameter values and the therefrom calculated imprinting factor and specific adsorption capacity of the MIP particles at binding saturation. For comparison, those from the adsorption data of the MIP and NIP particles with silicaMPS as the cores34 are also presented together. The MIP particles in this work show significantly higher imprinting factor and more than 3 times higher specific rebinding capacity than the silica-MPS-based MIP particles, thus confirming remarkable enhancement of the imprinting effect when the supports were replaced by the silicaCOOH particles. The ratio of the dissociate constant Kd for a NIP to that for the corresponding MIP also reflects the rebinding specificity of the MIP. As seen from Table 1, the Kd ratio in this work is about twice that obtained from the previous work, hence further verifying the great improvement of the imprinting efficiency. In comparison with the silica-MPS counterparts, the silica-COOH particles in this work are 10% smaller and hence cause equal increase in specific
external surface area. Although this difference may result in a slight increase of protein binding to the corresponding MIP particles, the above disparity in the imprinting effect of the core-shell-imprinted particles can be principally attributed to different surface chemistries of the internal core materials. It may be explained as follows. First, during the preassembly before the polymerization, although the silica-MPS particles could enrich the Lys template from the prepolymerization solution through the residual surface silanol groups, the silica-COOH particles would capture more Lys via the protruding carboxylic groups based on electrostatic interactions. This supposition is supported by the measured Lys binding capacities in the buffer of pH 7 with an initial concentration of 0.4 mg/mL, i.e., 39.5 mg/g for the silica-COOH particles versus 17.7 mg/g for the silica-MPS particles. Second, while surface graft polymerization for imprinting, the carboxylic groups, which stick out from the support surface via the spacer arms of an appropriate length (see Figure 1b), should be more advantageous to the cooperation with the functional and cross-linking monomers to create the imprinting sites. Both of the two points may be responsible for the remarkably enhanced imprinting results in terms of imprinting factor and specific adsorption capacity observed presently. The rebinding kinetics was further studied with an initial Lys concentration of 0.4 mg/mL. Both the MIP and NIP particles reached the equilibrium adsorption within 5 min (Figure S-3 in the Supporting Information), similarly to the results observed in ref 34. In contrast, the protein-imprinted nanoparticles reported by other researchers often required hours for achieving the adsorption plateau.26,27,30,31 Such strikingly fast kinetics is consistent with the superthin shell imprinted layers generated over the silica nanocores. Rebinding Specificity. Besides the template Lys, four other proteins with a wide range of isoelectric point (pI) and molecular weight (MW) were chosen as substrates to test the rebinding selectivity of the MIP particles. The pI and MW of all the proteins studied are Lys (MW 14K, pI 11.1), cytochrome c (Cyt c, MW 12.4K, pI 10.2), ribonuclease A (RNase A, MW13.7K, pI 9.6), bovine hemoglobin (Hb, MW 64.5K, pI 6.8), and bovine serum albumin (BSA, MW 66K, pI 4.8). Figure 4 shows the rebinding capacities of the MIP and NIP particles for these proteins with a feed concentration of 0.4 mg/mL. Obviously, the MIP particles exhibited much higher imprinting factor and specific rebinding capacity toward Lys than those toward the nontemplate proteins studied, verifying the specific recognition toward the print protein. With Cyt c as the competitor, the binary protein competitive adsorption experiments further confirmed the rebinding selectivity for Lys against Cyt c (see Figure 5). Most importantly, both the imprinting factor (2.12) and specific rebinding capacity (6.2 mg/g) for Lys herein observed are 1434
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Table 1. Parameter Values in the Langmuir Model and Therefrom Calculated Imprinting Factor and Specific Rebinding Capacity
a
qm (mg/g)
Kd (mg/mL)
qm,MIP/qm,NIP
qm,MIP - qm,NIP (mg/g)
Kd,NIP/Kd,MIP
MIPa
19.54 ( 0.55
0.041 ( 0.008
1.55
6.97
3.90
NIPa
12.57 ( 1.02
0.160 ( 0.047
MIPb
11.30 ( 0.40
0.049 ( 0.012
1.23
2.1
1.96
NIPb
9.20 ( 0.30
0.096 ( 0.015
With silica-COOH particles as cores. b With silica-MPS particles as cores.
Figure 4. Rebinding amounts of different proteins on the Lys-imprinted and nonimprinted particles. Adsorption conditions: V = 1.5 mL, m = 7.0 mg, Ci = 0.4 mg/mL, CNaCl = 40 mM, time 1 h, temperature 25 °C, Tris buffer (10 mM, pH 7.0). The imprinting factors are indicated above the bars. The binding of BSA was too little to be detected.
Figure 5. Competitive rebinding of Lys and Cyt c to the imprinted and nonimprinted particles. Adsorption conditions: V = 1.5 mL, m = 7.0 mg, Ci,Lys = Ci,Cyt c = 0.2 mg/mL, CNaCl = 40 mM, time 1 h, temperature 25 °C, Tris buffer (10 mM, pH 7.0). The imprinting factors are indicated above the bars.
remarkably increased in comparison with those (1.46 and 2.5 mg/g) achieved on the previous silica-MPS-based MIPs.34 This substantially confirms the imprinting enhancement with the newly designed surface modification for the core materials employed in this work. The recognition properties of the silica-COOH-based imprinted particles are comparable to those of the magnetic Lys-imprinted nanoparticles reported recently by Gai et al.30 with reference to imprinting factor and specific adsorption capacity. However, some measures may be taken to further enhance the imprinting effect. One possible approach is to adequately thicken the imprinted layers for sufficiently engulfing the template protein preadsorped onto the
Figure 6. Influence of the regeneration cycles on Lys adsorption to the imprinted particles and nonimprinted particles. Regeneration was performed by washing with 0.5 M NaCl solution. Adsorption conditions: V = 1.5 mL, m = 7.0 mg, Ci = 0.4 mg/mL, CNaCl = 40 mM, time 1 h, temperature 25 °C, Tris buffer (10 mM, pH 7.0). The imprinting factors are indicated above the bars.
support surface. This may be achieved via regulating the polymerization conditions like monomer concentration in a certain range. As for the Lys-imprinted nanoparticles reported by Jing et al.,31 the measured specific adsorption capacity was extraordinary high (∼100 mg/g) and even approached the Lys amount added while fabricating the imprinted particles (∼120 mg/g). This seems unreasonable, disagreeing with the general fact that the yield in effective imprints relative to the amount of imprint molecule used is quite low, especially when noncovalent imprinting method is adopted.1 Regeneration and Reproducibility. The regeneration feature is another important property of a protein MIP, considering its repeated utilization, the template recovery during the synthesis process, and target protein desorption when used as a separation medium. Unfortunately, until now fewer efforts have been made on this issue, probably due to the relatively harsh template-removing conditions often required for the preparation of the MIPs, e.g., extensive washing with SDS-acetic acid solution. As for the surface protein imprinting approach with preimmobilized template via forming an imine linkage, several groups10,13,17,27,33 have adopted this method, but none examined the repeated use of the MIPs, likely due to the severe template removal or desorption by time-consuming washing with oxalic acid or NaOH solution. In this work, however, the template removal during the MIP synthesis was achieved simply by washing with 0.5 M NaCl solution. Also, the regeneration of MIP particles after rebinding can be performed by the same way. Thanks to such a mild washing condition, the rebinding properties of the MIP particles remained almost unaffected after three cycles of adsorption-desorption, as can be seen from Figure 6. These results can be explained by the fact that the template molecules interact physically with the carboxyl groups attached on the support surface 1435
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Analytical Chemistry during either imprinting or rebinding, and also, very thin are the imprinted layers formed whereon.
’ CONCLUSIONS We have demonstrated the feasibility for the recognition enhancement of surface protein-imprinted nanoparticles with a core-shell structure through preimmobilization of noncovalent template sorption groups on the core surface. Surface imprinting of Lys over silica nanoparticles was studied for proof of this strategy. The introduction of carboxylic groups, which can electrostatically interact with the Lys molecules, on the support surface can significantly improve the imprinting effect of the resulting core-shell MIPs, much like the covalent template immobilization approach reported previously. Furthermore, it can endow the MIPs with the unique advantage of easy template removal or desorption, in contrast to the method based on covalent template linking. For imprinting of a target protein with this concept, the physical sorption groups to be attached on the support surface may be chosen based on a wide range of other noncovalent interactions with the imprint protein, e.g., hydrogen bonding, metal coordination, hydrophobic force, and chargetransfer complexing interaction. Work on this direction is already in progress and will be communicated in due course. ’ ASSOCIATED CONTENT
bS
Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Phone: þ86 22 23501443. Fax: þ86 22 23501443. E-mail: gqfu@ nankai.edu.cn.
’ ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Nos. 21074061 and 20574038) and the Natural Science Foundation of Tianjin (No. 09JCYBJC02900). ’ REFERENCES
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