Highly Porous Polymer Monolith Immobilized with Aptamer (RNA

Dec 30, 2015 - An aptamer immobilized tentacle-type monolith is prepared for lysozyme purification with excellent selectivity and high adsorbing capac...
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Highly Porous Polymer Monolith Immobilized with Aptamer (RNA) Anchored Grafted Tentacles and Its Potential for the Purification of Lysozyme Kaifeng Du,* Min Yang, Qi Zhang, and Shunmin Dan Department of Pharmaceutical & Biological Engineering, School of Chemical Engineering, Sichuan University, Chengdu 610065, P. R. China ABSTRACT: An aptamer immobilized tentacle-type monolith is prepared for lysozyme purification with excellent selectivity and high adsorbing capacity. In this study, the aptamer immobilized glycidyl methacrylate (GMA) serves as monomer to be grown into long polymer chains on a highly porous monolith by a series of reactions. This tentacle grafting gives rise to a sharp increase of aptamer coverage density and realizes the multilayer adsorption for protein. The dynamic adsorption capacity of the grafted monolith reaches 68.22 and 77.07 mg mL−1 for lysozyme at the breakthrough of 10% and 50%, respectively, up to approximately 10-fold higher than the ungrafted one. Despite the high adsorption capacity, the novel aptamer anchored tentacle grafted monolith separates perfectly lysozyme from the protein mixture, indicating the excellent adsorptive selectivity. By taking the advantages of high capacity and excellent selectivity, the affinity-grafted monolith is applied successfully to purify lysozyme from the diluted chicken egg white solution.

1. INTRODUCTION Aptamer is a single-stranded nucleic acid (RNA or DNA molecule) that can be artificially screened out by SELEX (systematic evolution of ligands by exponential enrichment). Ever since the discovery in the 1990s, it has shown great potential and multitudinous applications in the fields of biosensor, molecular interaction studies, and therapeutic drugs.1−4 It is well-known that the aptamers can identify specifically diverse target molecules (proteins and single organic small molecules, such as ATP, or amino acids), depending on their unique base sequence to bind highly specific structures, from huge complex molecules.5−7 Additionally, their small molecular weight and high molecular-density make them superior to traditional protein tools.8 Despite the fact that both RNA and DNA aptamers can form secondary structures using the classical Watson−Crick model, the RNA tertiary structure is of more diversity and thermodynamically stability as it harnesses the noncanonical base interaction.9−11 In contrast, DNA lacks the 2-hydroxyl functionality, thus reducing the opportunity for internal and intermolecular hydrogen bonding. Recently, the aptamers started to be used in the field of separation due to their high selectivity to the target molecules.8−10 Several groups presented the application of aptamer-based chromatography and proved the effectiveness of the novel affinity stationary phase for high performance separation. However, the current aptamer-based stationary phases often possess the disadvantage of low loading capacity, which hinders the practical separation application for both laboratory and preparative scale.8,12 In order to improve the adsorbing capacity, a common strategy is to increase the density of ligands by enlarging available inner surface area of support.13,14 Historically, decreasing the channel size and filling the hydrogel within support have been applied to increase the specific surface area of adsorbent.15,16 Unexpectedly, reducing the channels size often gives rise to high pressure drop along © 2015 American Chemical Society

with serious diffusion resistance, thus undoubtedly compromising separation efficiency.17,18 A possible alternative to resolve the problem is grafting the functional polymer tentacles on the support.19−21 With such modification, each grafted tentacle has the ability to link a certain amount of aptamers by a series of chemical reactions. During the chromatography process, these tentacle-type polymer chains with anchored aptamers would stretch outward from the pore surface to form a multilayer adsorption zone for high protein binding capacity. Based on this grafting strategy, we attempted to graft the apatmers anchored tentacles on pore surface of monolith by a series of chemical reactions and then evaluate its chromatographic performance by using lysozyme as probe. In the study, we chose the customized monolith with macropores as chromatography support. The large macroporosity is vital since it can provide enough space for the expansion of the grafted tentacles from the inner pore surface. For the adsorption evaluation, lysozyme was applied to evaluate the adsorption performance of the proposed tentacles grafted monolith. By taking advantages of macropores, tentacle-type polymer chains and aptamer ligands, the current research is hopeful to provide a novel strategy for fabricating the novel affinity monolith with high protein adsorption capacity.

2. EXPERIMENTAL SECTION 2.1. Materials. Glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) were bought from Yuanji (Shanghai, China). Benzoyl peroxide (BPO) (95%) was purchased from Damao (Tianjin, China) and recrystallized before use. The CaCO3 granules with an average size of 1.05 Received: Revised: Accepted: Published: 499

July 29, 2015 November 21, 2015 December 30, 2015 December 30, 2015 DOI: 10.1021/acs.iecr.5b02793 Ind. Eng. Chem. Res. 2016, 55, 499−504

Article

Industrial & Engineering Chemistry Research μm and a density of 2.70 g mL−1 were provided by Lihe (Tianjin, China). Lysozyme, cytochrome c, hemoglobin, and transferring were purchased from Sigma (St. Louis, MO, USA). The aptamer noted as 5-NH2(CH2)8GGUUGUGAAGAUUGGGAGCGUCGUGGCUAC-3 was provided by Midland Certified Reagent Company (Midland, TX, USA). All the other reagents, including dodecanol, cyclohexanol, D-glucosamines, methanol, ammonium ceric nitrate (ACN), and sodium periodate, were of analytical level and purchased from local resource. 2.2. Tentacles Grafted Porous Monolith. Macroporous monolith was fabricated by using template-assisted bulk polymerization with organic phase separation. During the preparation process, solid CaCO3 granules and inert solvent served as coporogen, which was similar to the previous report.13 Prior to the tentacles grafting, the glucose-anchored GMA monomer was prepared and denoted as gGMA. That is, 7 g of D-glucosamine and 4 g of GMA was dissolved together into 60 mL of methanol. With stirring at 50 °C for 2 h, the epoxy groups on GMA molecules were reacted completely with Dglucosamine, and the resultant was the glucose-anchored GMA (gGMA). The gGMA monomers were then grafted chemically on the pore surface of monolith. To perform the tentacles grafting, the monolith was activated with 0.1 M ammonium ceric nitrate (ACN) in 1 M HNO3 at 60 °C for 4 h. Following, the gGMA was pumped through the activated monolith at 40 °C for 30 min to initiate the radical polymerization. After the completed grafting reaction, the tentacles grafted monolith was obtained and ready for the next step. 2.3. Aptamer Immobilized Tentacles Monolith. Before the aptamer immobilization, the tentacles grafted monolith was flushed successively with 0.1 M sodium periodate solution for 20 h by the chromatography pump, which could oxidize completely the glucose groups on polymer chains into aldehyde terminals. The complete oxidization of glucose is a key factor to guarantee the reproduction of this procedure. Meanwhile, 0.2 M aptamer solution (50 mM Tris-HCl + 20 mM KCl + 600 mM NaCl, pH 7.4) was heated to 90 °C for 3 min and then dropped into room temperature. This treatment can reconstruct the aptamer into the expected secondary structure. Thereafter, the reconstructed aptamer contained solution was pumped through the monolith by using a chromatography pump at 50 °C and flow rate of 0.5 mL min−1 for 2.5 h to realize the aptamer immobilization. Finally, the affinity tentacles grafted monolith was prepared after washing the unbound aptamers from the monolith. The converge density of aptamers anchored on monolith was determined by weight balance according to the concentration decrease before and after aptamers immobilization. Meanwhile, the ungrafted monolith was also modified with the aptamers and compared it with the grafted one to highlight the role of tentacle grafting on the protein adsorption. In brief, 0.2 M D-glucosamine in methanol was pumped through the ungrafted monolith with epoxy groups at 50 °C for 2 h, which led to the immobilization of D-glucosamine on the monolith. Then, the immobilized glucoses were oxidized into aldehyde groups and further reacted with the reconstructed aptamers, as described above. By this reaction, the aptamer immobilized ungrafted monolith was obtained and ready for use. 2.4. Physical Characterization. Mercury injection method was applied to examine the macroporous size distribution of the prepared porous monoliths. The specific surface area of monolith was determined by NOVA 2000 porosimeter

(Quantachrome, USA). The pore size distribution was analyzed by mercury porosimeter (Quantanchrome Corporation, USA). The flow hydrodynamic behavior of monoliths was studied by analyzing the variation of back pressure along with flow rate of varied mobile phase, which were measured by Ä KTA Explorer 100 system installed with the Unicorn 4.1 software (Amersham Biosciences, Uppsala, Sweden). 2.5. Permeability Measurement. In HPLC, the hydraulic permeability was determined by measuring the pressure drop across the column as a function of flow rate of mobile phase. The hydraulic permeability is often used to evaluate the physical characteristics of the chromatography support and then guides its separation operation. The hydraulic permeability was calculated by the Darcy’s equation as follows.

Δp uμ = L B0

(1)

where Δp represents back pressure (Pa), u is the superficial velocity (m/s), L is the column length (m), B0 is the hydraulic permeability (m2), and μ is the mobile phase viscosity (Pa s). In addition, the mean porous diameter (d, nm) of monolith can also be estimated with the hydraulic permeability from the following equations: d=

32 × B0 ε

(2)

and ε=

Ve − Vc Vm

(3)

where ε is the porosity and determined from the elution volume (Ve, m3) of unretained component, system volume (Vc, m3), and the volume (Vm, m3) of the monolith. 2.6. Equilibrium Adsorption Experiments. The adsorption isotherms of lysozyme were performed on the prepared porous monolith via the protein breakthrough experiments. In brief, a group of 300 mL of lysozyme solution at different concentrations (0.1−1.2 mg mL−1) was circulated within both the monolith through a peristaltic pump for 12 h at room temperature, respectively. Then, the saturated adsorption capacity of the monolith was determined by analyzing the lysozyme concentration before and after the adsorption operation. The equilibrium adsorption capacity was calculated based on the weight balance. Finally, fitting these equilibrium data into Langmuir model (eq 4) gave the isotherm curves and the corresponding static adsorbing capacities and dissociation constants. Meanwhile, 20 mM Tris buffers were also prepared with 20 and 35 mM NaCl to explore the role of ion strength on the protein equilibrium adsorption. q c q= m kd + c (4) where q is the adsorbed lysozyme concentration, c is the free lysozyme concentration in equilibrium with q, qm and kd are the static adsorbing capacity and dissociation constant, respectively. 2.7. Breakthrough Measurement. Frontal analysis experiments were performed to determine the breakthrough behavior of both the grafted and ungrafted monoliths. Before the measurements, the monoliths were equilibrated with 20 mM Tris buffer until the absorbance of the outlet stream was stable. Then, 0.5 mg mL−1 lysozyme in 20 mM Tris buffer was pumped through the monolith at a defined flow rate and a UV 500

DOI: 10.1021/acs.iecr.5b02793 Ind. Eng. Chem. Res. 2016, 55, 499−504

Article

Industrial & Engineering Chemistry Research

Figure 1. Schematic illustration of the preparation process for the aptamer anchored tentacles grafting monolith.

selectivity, the grafted long polymer chains are expected to realize the multilayer adsorption for high protein adsorption capacity by expanding the adsorption zone from the limited pore surface into the inner pore volume. According to the grafting mechanism, the polymer chain length and density of the grafted tentacles would change the porous structure of monolith. Given the strong relationship between porous structure and chromatographic performance of support, it is necessary to explore the effect of tentacle grafting on the porous structure of monolith. To elucidate this point, varied physical characterizations (mercury intrusion analysis, N2 adsorption, pulse chromatography and flow hydrodynamics) were performed on the grafted and ungrafted monoliths. Figure 2 and Table 1 show the pore size distribution of the monoliths

detector at 280 nm monitored the outlet effluent. After the breakthrough was completed, the monolith was eluted with 20 mM Tris buffer plus 1 M NaCl and equilibrated with 20 mM Tris buffer for the next operation. The dynamic binding capacity was determined from the breakthrough curve and the following equation: qx =

c0(Vx − V0) Vb

(5)

where qx is the dynamic binding capacity at x% breakthrough, c0 is feed lyzolyme concentration, and Vb, Vx, and V0 are monolith volume, feed lysozyme solution volume at x% breakthrough point, and the void volume of monolith, respectively. 2.8. Purification of Lysozyme from Chicken Egg White. The chicken egg white was collected from fresh eggs and diluted to a quarter of the original sample with 20 mM Tris buffer. The diluted egg white solution was mechanically stirred in an ice bath for 4 h, and then centrifuged at 4 °C and 12 000 rpm for 20 min. After the centrifugation, the diluted lysozyme solution was applied for the chromatography evaluation. Prior to the chromatography operation, the aptamers anchored tentacles grafted monolith was equilibrated with 20 mM Tris buffer. Then, 50 μL of lysozyme solution was injected into the monolith at the rate of 0.5 mL min−1 through HPLC system. After 12 min, the monolith was immediately flushed with 20 mM Tris buffer plus 1 M NaCl to elute the lysozyme from the monolith.

3. RESULTS AND DISCUSSION In the study, tentacles polymer chains with a plurality of aptamer groups were grown from the inner pore surface of highly porous monolith and evaluated for lysozyme adsorption. Figure 1 shows the grafting strategy and the adsorption mechanism for high protein adsorption capacity in details. That is, the amine groups of D-glucosamine were reacted with the epoxy groups of GMA to generate the glucose anchored GMA (gGMA). The gGMA resultant serving as new monomer was grafted from the pore surface of monolith by cerium induced free radical polymerization. After the tentacles grafting reaction, the anchored glucose on the polymer tentacles was oxidized into the aldehyde groups for the aptamer immobilization. Together with the aptamer immobilization for high adsorption

Figure 2. Pore size distributions of the ungrafted (○) and grafted (□) monoliths.

before and after the tentacle grafting. As shown here, both monoliths shared very similar appearance of pore size distribution curves, at which the highest pore volume peaks were centered at about 2 μm in pore size. It might be an indication that the surface grafting has no evidently effect on the macropore channels by the evaluation of mercury intrusion analysis. That is, the prepared monolith still possessed the interconnected macropores and had the ability to allow the mobile phase to pass through the support with perfusion flow, 501

DOI: 10.1021/acs.iecr.5b02793 Ind. Eng. Chem. Res. 2016, 55, 499−504

Article

Industrial & Engineering Chemistry Research

mobile phase was forced to pass through the monolith during the chromatography process, the grafted tentacles bearing the aptamers became charged and moved more freely within the pore cavity. The repellent interaction by the charged groups then caused the adjacent tentacles to be far away from each other as possible. Accordingly, the pore size was reduced by the tentacles expansion from the pore surface and then led to the steric hindrance for low column permeability. In general, the moderate expansion of grafted tentacles within porous channels can expose fully the anchored adsorption sites and makes the monolith possible for high protein adsorption capacity. Breakthrough analysis is an important technique for evaluating the pore transport and adsorption characteristics of monolith. It is more practical because the actual chromatographic process might be more really reflected by its breakthrough behavior. In this context, the breakthrough experiments were performed on the grafted and ungrafted monoliths. Figure 4 shows the results of a series of

Table 1. Physical Characteristics of Both the Grafted and Ungrafted Monoliths (4.6 × 50 mm) samples bed permeabilitya B (×10−14 m2) mean pore sizeb (nm) macropore sizec (nm) porosity (%) specific surface area (m2 g−1)

ungrafted monolith

grafted monolith

5.98

2.54

1642 685−2600 0.71 29

998 635−2200 0.68 32

a Bed permeability was obtained by using 20 mM Tris buffer(pH 7.4) as mobile phase. bMean pore size was calculated from eq 2 in section 2.4.1. cMacropore size was determined by mercury intrusion measurement.

as the previous report.13 Differently, the porosity decreased by 4.2% and the specific surface area increased by 10.3% after the tentacle grafting polymerization, as shown in Table 1. The increased specific surface area might be originated from the tentacle grafting and is expected to contribute to high adsorption capacity for protein purification. According to the mercury porosimetry, there was no significant difference in pore size distribution between the ungrafted and grafted monoliths. While suited for the evaluation of the monolith in the dry state, the mercury intrusion method cannot reflect the contribution of the hydrated tentacles polymer chains on the apparent pore size. It can be explained by that the grafted tentacles in the dry state existed only as a dehydrated flat layer and then led to little contribution to the reduction of pore size. However, the grafted tentacles would expand more or less in pore volume during the chromatography operation, which depended upon the choice of solvent.13 Given this, we performed further the flow hydrodynamics measurement on the grafted monolith, which is shown in Figure 3. From the flow hydrodynamics curve, it

Figure 4. Breakthrough curves by using the ungrafted monolith (left) and the grafted monolith (right) at varied flow rates.

Figure 3. Relationship between back pressure and flow rate for the ungrafted monolith (○) and the grafted monolith (□).

breakthrough curves by using lysozyme solution as mobile phase under different flow rates (0.5, 1.0, and 1.5 mL min−1). It is obvious that the breakthrough points and shapes of both the grafted and ungrafted monoliths changed less with an increase of flow rate. Differently, the slopes of the breakthrough curves of the grafted monolith were larger than the ungrafted one. The long tail of breakthrough curve demonstrated that the grafted tentacles resulted in higher mass transfer resistance on the monolith when the mobile phase passed through the grafted pore channels. Moreover, to quantitatively elucidate the advantage of grafting, several important parameters were calculated and listed in Table 2. It is found that the tentacles grafting contributed effectively the monolith to high coverage density and further to large static and dynamic binding capacities. With the tentacle grafting, the coverage density of aptamer increased from 1.461 to 25.356 μmol mL−1. More Table 2. Static and Dynamic Adsorbing Capacities of Lysozyme on the Grafted and the Ungrafted Monoliths

reveals that the back pressure on the grafted monolith is significantly higher in comparison to the ungrafted one in the tested flow rates. To elucidate quantitatively this point, the column permeability was calculated from Darcy’s law (eq 1), which was tabulated in Table 1. It can be seen that the grafted monolith possessed the column permeability of 2.54 × 10−14 m2, which was about 2.35-fold reduction to the ungrafted ones (5.98 × 10−14 m2). The reduction of column permeability can be ascribed to the polyelectrolyte theory. That is, when the

samples coverage density (μmol mL−1) adsorption equilibrium capacity (mg mL−1) dynamic adsorption capacity (mg mL−1)

502

ungrafted monolith

grafted monolith

1.461 15.12

25.356 101.55

6.51 (10%) 8.64 (10%)

68.22 (10%) 77.07 (10%)

DOI: 10.1021/acs.iecr.5b02793 Ind. Eng. Chem. Res. 2016, 55, 499−504

Article

Industrial & Engineering Chemistry Research

The essential feature of the prepared monolith lies in its specificity of the aptamer ligands to lysozyme. To confirm this, several proteins (cytochrome c, hemoglobin, transferring) with different isoelectric points were mixed with lysozyme, respectively, and then separated by the grafted monolith. The corresponding chromatographic behavior is shown in Figure 6.

remarkably, the dynamic binding capacities reached about 68.22 and 77.07 mg mL−1 for lysozyme at 10% and 50% breakthrough points on the grafted monolith, respectively, which was approximately 10-fold higher over the ungrafted one. Obviously, the tentacle grafting was considered as the key factor for the increase of protein adsorption capacity since the protein multilayer adsorption was realized by expanding the adsorption zone from the limited pore surface to the internal pore volume. This observation agrees with the works done by previous researchers.13 Static adsorption measurements were carried out to evaluate the grafted and ungrafted monoliths. Figure 5 shows the

Figure 6. Chromatographic separations of lysozyme and different proteins (cytochrome c, hemoglobin, transferring) on the grafted monolith.

As seen here, all the nontarget proteins were eluted out from the column after the sample injection, while the lysozyme was selectively captured. The adsorbed lysozyme was eluted only when the mobile phase was shifted into the elution buffer. Since the cytochrome c has the similar basic pI with lysozyme, the high performance separation between cytochrome c and lysozyme confirms that the affinity interaction plays an important role in this retention rather than the electrostatic interaction. To evaluate the practical application, the prepared tentacle grafted monolith with aptamer ligands was applied to screen lysozyme from the rough extraction of chicken egg white. The chromatography operation was described in Section 2.4.4, which included three steps of equilibrium, adsorption and elution. Figure 7 shows the chromatogram of lysozyme purification from chicken egg white extraction. It reveals that the nontarget proteins in chicken egg white had no retention on the aptamer anchored tentacles grafted monolith, and eluted out as the first peak. When the mobile phase was shifted to the elution solution (20 mM L−1 tris+1 M NaCl), another peak

Figure 5. Effect of ion strength (□, 0 mM NaCl; ○, 20 mM NaCl; Δ, 35 mM NaCl) on adsorption isotherm curves on the ungrafted monolith (left) and the grafted monolith (right).

adsorption isotherms on both monoliths under different NaCl concentrations from 0 to 35 mM. Fitting these data gave the adsorption equilibrium constants, which were tabulated in Table 3. It reveals from Figure 5 and Table 3 that the static Table 3. Effect of NaCl Concentrations on qm and Kd of the Grafted and Ungrafted Monoliths ungrafted monolith

grafted monolith

NaCl (mmol L−1)

qm

Kd

qm

Kd

0 20 35

15.09 13.26 11.31

0.096 0.178 0.287

91.14 82.89 64.44

0.064 0.167 0.242

adsorption capacity reduced regularly and the dissociation constants increased with an increase of NaCl concentrations. Additionally, the grafted monolith enjoyed larger adsorption capacity and relatively lower dissociation constant for lysozyme adsorption when the NaCl concentration was same. Such as, the static adsorption capacity of the grafted monolith reached about 91.14 mg mL−1 for lysozyme, which was 6 fold larger than the ungrafted one (15.09 mg mL−1). This somehow confirmed the assumption that the multilayer adsorption for lysozyme was realized on the grafted monolith. Moreover, the adsorption capacity reduced and the dissociation constant increased regularly with the elevation of ion strength despite the tentacle grafting. This result can be explained by that increasing ion strength weakens the interaction between the proteins and the aptamer groups and then compromises the protein adsorption capacity.

Figure 7. Separation of lysozyme from chicken egg white on the grafted monolith. 503

DOI: 10.1021/acs.iecr.5b02793 Ind. Eng. Chem. Res. 2016, 55, 499−504

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Industrial & Engineering Chemistry Research

(9) Potty, A. S. R.; Kourentzia, K.; Fang, H.; Schuck, P.; Willson, R. C. Biophysical characterization of DNA and RNA aptamer interactions with hen egg lysozyme. Int. J. Biol. Macromol. 2011, 48, 392. (10) Orozco, J.; Campuzano, S.; Kagan, D.; Zhou, M.; Gao, W.; Wang, J. Dynamic Isolation and Unloading of Target Proteins by Aptamer-Modified Microtransporters. Anal. Chem. 2011, 83, 7962. (11) Han, B.; Zhao, C.; Yin, J.; Wang, H. High performance aptamer affinity chromatography for single-step selective extraction and screening of basic protein lysozyme. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2012, 903, 112. (12) Musumeci, D.; Montesarchio, D. Polyvalent nucleic acid aptamers and modulation of their activity: a focus on the thrombin binding aptamer. Pharmacol. Ther. 2012, 136, 202. (13) Du, K.; Yang, D.; Sun, Y. Fabrication of high-permeability and high-capacity monolith for protein chromatography. J. Chromatogr. A 2007, 1163, 212. (14) Jain, P.; Sun, L.; Dai, J.; Baker, G. L.; Bruening, M. L. HighCapacity Purification of His-tagged Proteins by Affinity Membranes Containing Functionalized Polymer Brushes. Biomacromolecules 2007, 8, 3102. (15) Du, K.; Yan, M.; Song, H.; Zhang, Y. Synthesis of Bimodal Porous Titania Beads and Their Potential in Liquid Chromatography. Ind. Eng. Chem. Res. 2011, 50, 6101. (16) Leonard, M. New packing materials for protein chromatography. J. Chromatogr., Biomed. Appl. 1997, 699, 3. (17) Lungfiel, K.; Seubert, A. Varying the porous structure of polystyrene/ divinylbenzene beads prepared by Ugelstads activated swelling technique and examining its reversed phase HPLC properties. J. Chromatogr. A 2014, 1358, 117. (18) Gokmen, M. T.; Camp, W. V.; Colver, P. J.; Bon, S. A. F.; Du Prez, F. E. Fabrication of Porous “Clickable” Polymer Beads and Rods through Generation of High Internal Phase Emulsion (HIPE) Droplets in a Simple Microfluidic Device. Macromolecules 2009, 42, 9289. (19) Chenette, H. C.S.; Robinson, J. R.; Hobley, E.; Husson, S. M. Development of high-productivity, strong cation-exchange adsorbers for protein capture by graft polymerization from membranes with different pore sizes. J. Membr. Sci. 2012, 423, 43. (20) Singh, N. K.; Dsouza, R. N.; Grasselli, M.; Fernández-Lahore, M. High capacity cryogel-type adsorbents for protein purification. J. Chromatogr. A 2014, 1355, 143. (21) Xu, F.; Geiger, J. H.; Baker, G. L.; Bruening, M. L. Polymer Brush-Modified Magnetic Nanoparticles for His-Tagged Protein Purification. Langmuir 2011, 27, 3106. (22) Bayramoglu, G.; Tekinay, T.; Ozalp, V. C.; Arica, M. Y. Fibrous polymer grafted magnetic chitosan beads with strong poly(cationexchange) groups for single step purification of lysozyme. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2015, 990, 84.

appeared and was designated to lysozyme. The result confirms that the anchored aptamer as affinity ligand could recognize specifically and purify lysozyme from chicken egg extraction. The excellent specificity together with the high adsorption capacity make the aptamers anchored tentacles grafted monolith has great potential in the practical protein purification.

4. CONCLUSIONS A novel aptamers anchored tentacles grafted monolith was developed by grafting glucose-GMA monomers together with aptamers immobilization via a series of chemical reactions. With the flexible grafted tentacles, not only the porous monolith has reserved relatively high permeability for fast mass transfer but also these grafted tentacles endowed the monolith with high aptamer coverage density, as a result of high protein adsorption capacity. The high adsorption capacity contributed to the occurrence of multilayer adsorption for lysozyme, in which the grafted tentacles expanded the adsorption zone from the limited pore surface to the inner pore volume. Except for high adsorption capacity and excellent permeability, the affinity tentacle grafted monolith exhibits excellent specificity for the lysozyme adsorption. Finally, the practical sample separation demonstrated that the novel affinity monolith has a great potential for identify and purify lysozyme from the complex biological mixture.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +86-28-85405221. Fax: +8628-85405221. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was funded by Natural Science Foundation of China (21206097 and 21476144) and Science Foundation for The Excellent Youth Scholar of Sichuan University (2014SCU04A04).

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REFERENCES

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DOI: 10.1021/acs.iecr.5b02793 Ind. Eng. Chem. Res. 2016, 55, 499−504