- Email: [email protected]
Immobilization of BSA on ionic liquid functionalized magnetic Fe3O4 nanoparticles for use in surface imprinting strategy
Author’s Accepted Manuscript Immobilization of BSA on ionic liquid functionalized magnetic Fe3O4 nanoparticles for use in surface imprinting strategy Liwei Qian, Jiexuan Sun, Chen Hou, Jinfan Yang, Yongwei Li, Dan Lei, Miaoxiu Yang, Sufeng Zhang www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(17)30346-6 http://dx.doi.org/10.1016/j.talanta.2017.03.044 TAL17389
To appear in: Talanta Received date: 14 January 2017 Revised date: 5 March 2017 Accepted date: 16 March 2017 Cite this article as: Liwei Qian, Jiexuan Sun, Chen Hou, Jinfan Yang, Yongwei Li, Dan Lei, Miaoxiu Yang and Sufeng Zhang, Immobilization of BSA on ionic liquid functionalized magnetic Fe3O4 nanoparticles for use in surface imprinting strategy, Talanta, http://dx.doi.org/10.1016/j.talanta.2017.03.044 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Immobilization of BSA on ionic liquid functionalized magnetic Fe3O4 nanoparticles for use in surface imprinting strategy Liwei Qiana,b*, Jiexuan Suna, Chen Houa,b, JinfanYanga,b, Yongwei Lia,b, Dan Leia, Miaoxiu Yanga, Sufeng Zhanga, a
College of Bioresources Chemical and Materials Engineering, Shaanxi University of Science and Technology,
Xi’an 710021, China b
Key Laboratory of Pulp and Paper Science & Technology of Ministry of Education/Shandong Province, Qilu
University of Technology, Jinan, 250353, China
[email protected] (L. Qian), [email protected] (S. Zhang)
*Corresponding author. Tel.: +86 13669272714
ABSTRACT Combining template immobilization with surface imprinting technology is an effective strategy to overcome the difficulties associated with macromolecular template removal and to achieve high specific recognition ability. In this work, ionic liquid functionalized Fe3O4 nanoparticles were prepared via a simple two-step modification process and were used as substrate to immobilize bovine serum albumin (BSA). The zeta potential study revealed immobilization of BSA on the nanoparticles through multiple interactions, and the immobilization capacity was about nine times higher compared with that of bare Fe3O4. Subsequently, dopamine was utilized as functional monomer to prepare BSA surface imprinted nanoparticles. Fourier transform infrared spectroscopy, thermo-gravimetric analysis and transmission electron microscopy verified the successful preparation of BSA imprinted nanoparticles with core-shell structure. The influence of imprinted layer thickness on recognition ability of imprinted nanoparticles was investigated, and the results suggested that 20 nm was an optimum thickness to achieve the best recognition ability. The adsorption isotherm studies showed that the imprinted nanoparticles had a significantly higher adsorption capacity and stronger binding affinity than the non-imprinted ones. Furthermore, the selective as well as the competitive adsorption studies revealed higher selectivity and recognition ability of the
imprinted nanoparticles for BSA. Therefore, the proposed strategy is an effective way to obtain protein imprinted polymers with high adsorption capacity and good recognition ability, thus would be beneficial for the further development and application of protein imprinting technology. Key words: Protein imprinted polymers; Ionic liquids modification; Protein immobilization; Magnetic nanoparticles;
1. Introduction Molecularly imprinted polymers (MIPs), a kind of artificial synthetic material with highly selective recognition ability for template molecules, have drawn much attention in the past few years [1-2]. Owing to their good stability, resistance to harsh environments and selective recognition ability, MIPs for small molecular template have been successfully applied in a wide variety of fields including applications in biosensors [3], bioseparation [4], medical diagnostics [5], and drug delivery [6], etc. However, unlike small molecules, imprinting of macromolecules, especially for protein, enzymes, DNA, viruses and even cells poses a great challenge because of their huge size, structural complexity, and conformational flexibility [7-8]. To address these issues, a variety of ingenious imprinted strategies, such as epitope imprinting approach [9], surface imprinting approach [10] and imprinting using macromolecular functional monomer and cross-linker [11], etc., have been proposed. Among these approaches, the method that combines template immobilization with surface imprinting technology has been regarded as a potential strategy to effectively overcome the difficulties in macromolecular removal and rebinding [12-13]. This method allows creating thin-film like polymer structures containing imprinting cavities, located at or close to the surface of imprinted polymers, thereby facilitating the removal of template and enhance the specific recognition ability of MIPs. Hao et al. proposed an effective method to prepare surface imprinted nanoparticles via covalent immobilization of hemoglobin [14]. The obtained MIPs showed good specific recognition ability with an imprinting factor of 3.85, and the rapid adsorption equilibration time of MIPs demonstrated the advantage of surface imprinting. However, covalent immobilization method requires rather harsh conditions for template removal, which might affect imprinting stability. Compared with covalent immobilization, non-covalent attachment of protein to the surface of functionalized substrate is more promising in surface imprinting technology due to moderate elution conditions [15]. Nevertheless, adopting single binding pattern, like hydrogen binding or electrostatic binding, might lead to relatively low immobilization capacity. Therefore, adopting multiple binding pattern in non-covalent approach is a potential way to obtain substrate with high immobilization capacity. Based on the above reasons, substrate functionalized with ionic liquids (IL) group of imidazole type has drawn
worldwide attention due to their multiple interactions [16-18]. Recently, our group has reported a surface imprinted immunostimulating peptide on raspberry-like microsphere via non-covalent immobilization [19], and we have used IL functionalized substrate to immobilize template in the work. We found that due to the multiple functional groups of IL, peptide immobilization capacity reached to 22.4 mg g-1, five times higher than the substrate without IL modification. Therefore, immobilization of template using IL functionalized substrate in surface imprinting technology would facilitate preparation of MIPs with high capacity and selectivity. Controlling the thickness of imprinted layer is another critical factor in surface imprinting technology [20-21]. An overmuch thickness of imprinted layer causes embedding of template, while a too thin imprinted layer may create incomplete template-shape cavities. Both situations can reduce selectivity and recognition ability of MIPs. Thus, it is still a developmental trend in macromolecule imprinting to choose or utilize controllable polymeric monomer or method. Dopamine (DA) can self-polymerize to generate polydopamine (PDA) under weak alkaline solution at 25 ºC. By controlling DA concentration and reaction time during polymerization, an adjustable, biocompatible and hydrophilic imprinting layer can easily be obtained [22-23]. Wu et al. reported a series of thickness-tunable imprinting films in nanometer-scale via controlling polymerization time of DA [24] and found that the PDA imprinting film with an optimal thickness exhibited significant selectivity towards glycoprotein, indicating the importance of a template-size matched imprinted layer in MIPs. In order to obtain the MIPs with high adsorption properties and recognition ability, the surface imprinting technology with thickness control is combined with an IL-based template immobilization technology for the first time in this work. First of all, we prepared an IL functionalized Fe3O4 nanoparticle (Fe3O4@IL) via a simple two-step modification process, and utilized Fe3O4@IL nanoparticles to immobilize bovine serum albumin (BSA), which was subsequently imprinted via using DA as a functional monomer. The resulting magnetic nano-materials were characterized by transmission electron microscope (TEM), X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, and vibrating sample magnetometer (VSM). Meanwhile, the adsorption behavior of MIPs was investigated by isothermal rebinding and dynamic adsorption. Furthermore, specific recognition ability of MIPs was investigated using selective adsorption experiment, competitive adsorption experiment, and real sample test.
2. Material and methods 2.1. Materials Dopamine (DA), ferric chloride hexahydrate (FeCl3·6H2O), anhydrous sodium acetate (NaOAc), ethylene
glycol (EG), sodium citrate, bovine serum albumin (BSA), ovalbumin (OVA) and lysozyme (Lyz) were purchased from sigma (St. Louis, MO, USA). (3-Chloropropyl)trimethoxysilan (Cptms) and 1-Methylimidazole were obtained from was supplies by Acros Organics (Morris Plains, NJ, USA). All reagents were of at least analytical grade and used without further treatment. 2.2. Instrumentation Fourier transforms infrared (FTIR) spectra were acquired on a TENSOR27 FTIR spectrometer (Bruker). The samples were prepared by mixing the products with KBr and pressing into a compact pellet. Morphology and structure of the nanoparticles were observed using transmission electron microscopy (TEM, JEOL JEM-3010). The polymer content of these nanoparticles was determined by thermo-gravimetric analysis (TGA, Q50, TA instruments) in the temperature range of 100 to 700 oC, with a heating rate of 10 oC min-1 under nitrogen atmosphere. Room temperature magnetization isotherms were obtained using a vibrating sample magnetometer (VSM, LDJ9600). Zeta potential is measured by using a commercial laser light scattering spectrometer (Zetasizer Nano-ZS). The absorption spectra were measured using a UV spectrophotometer (Varian, Cary-1E). The crystal phase was collected by a PAN analytical X’pert Pro diffractometer (XRD, Almelo). Protein identification was performed using a Shimadzu LC-2010A series HPLC with an Xtimate SEC column (250 mm × 7.8 mm, 5 μm, 300 Å). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a DYY-6C electrophoresis system (Beijing Liuyi instrument plant). 2.3. Synthesis of Fe3O4 nanoparticles Fe3O4 nanoparticles were prepared using a previously reported solvothermal method with some modifications [25]. Briefly, FeCl3·6H2O (3.0 g) was taken in ethylene glycol (100 mL) containing NaOAc (4.8 g) and sodium citrate (0.7 g), and the mixture was heated with vigorous stirring to obtain a homogeneous solution. The solution was then transferred into a 200 mL Teflon reactor and heated at 200 ºC for 10 h, followed by cooling down to 25 ºC. The product was washed several times with deionized water and ethanol and finally, dried in a vacuum at 30 ºC overnight to obtain Fe3O4 nanoparticles. 2.4. Synthesis of chloro-functionalized Fe3O4 nanoparticles A mixture of Fe3O4 nanoparticles (1 g) in dry toluene (25 mL) was sonicated for 30 min, and 3-Cloropropyl trimethoxysilane (0.5 mL) was then added to the dispersed Fe3O4 nanoparticle in toluene. The reaction mixture was slowly heated to 115 ºC and then stirred at this temperature for 24 h. The obtained product was separated by an external magnet and washed three times with diethyl ether and CH2Cl2, followed by drying under vacuum. 2.5. Synthesis of Fe3O4@IL nanoparticles
1-Methylimidazole (0.2 mL) was added dropwise to chloro-functionalized Fe3O4 nanoparticles (0.5 g) in 250 mL ethanol at 60 ºC under a nitrogen atmosphere. Once the addition was complete (30 min), the mixture was stirred for 24 h. The product was separated by an external magnet, washed with ethanol, and finally, dried at 25 ºC in vacuum to obtain Fe3O4@IL nanoparticles. 2.6. Synthesis of Fe3O4@IL@MIP To prepare BSA surface imprinted magnetic nanoparticles, Fe3O4@IL nanoparticles (0.2 g) was first incubated in BSA Tris-HCl buffer (pH 8.0, 10 mM) solution at a concentration of 1.2 mg mL-1. After 3 h incubation, Fe3O4@IL nanoparticles were magnetically separated and washed with deionized water. Subsequently, Fe3O4@IL nanoparticles were dispersed in 100 mL of Tris-HCl buffer (pH 8.0, 10 mM) in a three-necked flask, and DA (0.1 g) was added to the solution with continuous mechanical stirring at 25 ºC for 4 h. The obtained product (designed Fe3O4@IL@MIP) was washed with NaCl solution (0.1 M) to remove the template protein until no adsorption was detected by UV-vis spectrophotometer at about 278 nm. The product was further washed with deionized water to remove NaCl and dried under vacuum. The non-imprinted polymers (designed Fe3O4@IL@NIP) were prepared using the same protocol as Fe3O4@IL@MIP but without the addition of BSA. 2.7. Adsorption experiments of Fe3O4@IL@MIP and Fe3O4@IL@NIP To investigate the adsorption kinetics of the adsorbent, 50 mg of Fe3O4@IL@MIP were suspended in 10 mL of BSA Tris-HCl buffer (pH 8.0, 10 mM) solution at a concentration of 1.0 mg mL-1. The mixture was incubated at regular time intervals from 5 min to 80 min at 25 ºC, and the polymers were magnetically separated from the solution. The concentration of BSA in residual solution was measured by UV-vis spectrophotometer. The binding tests were performed at room temperature (25 ºC) using BSA solutions of different initial concentrations, ranging from 0.1 to 1.2 mg mL-1. After 12 h incubation, which was sufficient to reach adsorption equilibrium, the polymers were magnetically separated from the solution, and the concentrations of solutions were measured by a UV-vis spectrophotometer at 278 nm. The adsorption capacities (Q) of the MIPs for the template protein BSA were calculated using the following formula: Q = (C0 - Ce)V/m, where C0 is the initial concentration of BSA (mg mL-1), Ce is the BSA concentration (mg mL-1) of the supernatant solution, V is the volume of the BSA solution (mL), and m is the mass of MIPs (g). To evaluate the specificity of MIPs toward the BSA template or template analogues, imprinting factor (IF) was used and calculated from the following equation: IF = QMIPs/QNIPs, where QMIPs and QNIPs are the adsorption capacity of the template or template analogues by MIPs and NIPs, respectively. To investigate the selectivity of the imprinted material, 50 mg of Fe3O4@IL@MIP nanoparticles were added to
a centrifuge tube containing 10 mL protein solution (template and template analogue separately) of concentration 1.0 mg mL-1 at 25 ºC and shaken for 12 h. After magnetic separation of Fe3O4@IL@MIP from the solution, the concentration of protein in the supernatant solution was measured using a UV spectrophotometer. The selectivity factor (β) is defined as the following equation: β = IFtemp/ IFana, where IFtemp and IFana are the imprinting factors for the template molecule and for the analogues, respectively. In the competitive adsorption experiments, 50 mg of Fe3O4@IL@MIP nanoparticles were conditioned in a 10 mL binary mixture solution (containing both the template and its analogue) at 25 ºC with each protein concentration of 1.0 mg mL-1. After 12 h incubation, Fe3O4@IL@MIP was magnetically separated from the solution, and the concentrations of template protein and analogue in the supernatant solution were measured using HPLC. The separation factor (α) was calculated to evaluate the separation ability of adsorbing material in a mixture of BSA and its analogues. The separation factor is defined by the following equation: α = QBSA/Qana, where QBSA and Qana are the binding capacities of BSA and its analogues, respectively. In order to further investigate the specific recognition ability of Fe3O4@IL@MIP, the tolerance concentration of BSA’s analogue in the competitive adsorption experiments was carried out. In this experiment, the concentration of BSA was fixed at 1.0 mg mL-1, while the concentration of its analogue was increased from 1.0 mg mL-1 until the α value was about 1. Other conditions were same as above competitive adsorption experiments. In the real sample test, bovine calf serum (BCS) was 10-fold diluted with phosphate buffer (10 mM, pH 8.0). About 50 mg of Fe3O4@IL@MIP was conditioned in the above serum solution at 25 ºC for 12 h. The particles were treated with phosphate buffer (10 mM, pH 8.0) containing 5 mM NaCl to remove the weakly adsorbed proteins and then with 0.1 M NaCl solution to elute the strongly adsorbed proteins. The elution process was continued until no characteristic peaks of these proteins showed in the UV region of the spectrum. The eluate for strongly adsorbed proteins was combined, desalted by using dialysis tubing with a molecular cut-off of 500, and then lyophilized. The obtained products were dissolved using phosphate buffer (10 mM, pH 8.0), and their concentrations were maintained at 1 mg mL-1. To separate and analyze the proteins, gel-electrophoresis was performed using SDS-PAGE (12% polyacrylamide separating gel and 5% polyacrylamide stacking gel); 20 μL of product solution was taken for running SDS-PAGE electrophoresis. 2.8. Computer simulation In order to study the immobilization mechanism of Fe3O4@IL, first-principles calculations based on density functional theory (DFT) were performed to investigate their hydrogen bond and π-π stack interaction (b3lyp/6-31g(d) was used as basis set) [26]. According to the previous report [27], we simplified the models of
Fe3O4@IL and BSA in order to facilitate the computer simulation. Among them, Fe3O4@IL could be simplified as 1-propyl-3-methylimidazole, while the functional groups in BSA could be replaced by CH3-X (X=SH, OH, SSCH3, COOH, NH2, C6H5). The binding energies (ΔE) of the complexes formed from 1-propyl-3-methylimidazole and functional groups in BSA were evaluated by the following equation:
ΔE E (complex) E ( IL) E ( FG) where E(IL) , E(FG) and E(complex) were the potential energies of 1-propyl-3-methylimidazole, functional groups in BSA, and their complex respectively.
3. Results and discussion 3.1 Production process of Fe3O4@IL@MIP Scheme 1 illustrates the synthesis of Fe3O4@IL@MIP, combining the surface imprinting technique and immobilized template strategy. Fe3O4 nanoparticles were first prepared according to a modified solvothermal method and subsequently, the nanoparticles were allowed to react with (3-chloropropyl)trimethoxysilane to obtain chloro-functionalized Fe3O4. In the next step, Fe3O4@IL with abundantly ionized imidazole groups were prepared via alkylation reaction, and BSA was easily immobilized through hydrogen bond, electrostatic, and π-π force interactions. Immobilization of template protein on supporter through non-covalent interactions not only overcomes the disadvantage of the harsh desorption condition for covalent template immobilization, but also possesses the advantage of better imprinting effect to some degree when compared with that achieved from the traditional free-template polymerization process. Finally, an appropriate thickness of PDA layer was coated on the surface of Fe3O4@IL-BSA complex, and Fe3O4@IL@MIP nanoparticles with recognition sites complementary to BSA were obtained after the removal of the embedded template protein by simple elution with NaCl solution.
Scheme 1. Schematic illustration of the synthesis Fe3O4@IL@MIP nanoparticles
3.2 Immobilization of BSA on Fe3O4@IL Since the immobilization process of the template protein BSA on Fe3O4@IL was crucial, their interaction mechanism should be investigated. In this work, we utilized Zeta potential to qualitatively analyze the multiple-interaction between Fe3O4@IL and BSA; meanwhile, we also investigated the influence of BSA concentration on immobilization capacity of Fe3O4@IL. Fig.1 shows that the Zeta potential value of Fe3O4@IL is 26.5 mV at pH 8.0 due to the presence of ionized imidazole group on the surface of Fe3O4@IL. After interacting with BSA (isoelectric point 4.7), Zeta potential value of Fe3O4@IL sharply decreases. The immobilization effect in this process might be dominated by electrostatic interaction. When BSA concentration is 0.4 mg mL-1, the exterior of the Fe3O4@IL appears nearly neutral. Further increasing the concentration of BSA, they could be still adsorbed on Fe3O4@IL, which should be due to hydrogen bond and π-π interaction, resulting in further decrease of Zeta potential value (Hydrogen bond and π-π interaction between Fe3O4@IL and BSA was further confirmed by computer simulation, and results were shown in Table S1 and Fig.S1). We also observed similar phenomenon in our previous research [28]. Finally, the Zeta potential value of Fe3O4@IL is fixed to -15.8 mV at the BSA concentration of 1.2 mg mL-1, when the maximal adsorption capacity is 36.8 mg g-1. This maximal adsorption capacity is significantly higher compared with that of the bare Fe3O4 (3.8 mg g-1) and chloro-functionalized Fe3O4 (5.1 mg g-1) nanoparticles at the same condition. Therefore, the above result demonstrates that Fe3O4@IL could preferably immobilize BSA via multiple interactions.
Fig. 1. Influence of BSA concentration on the Zeta potential and immobilization capacity of Fe3O4@IL at pH 8.0
3.3 Characterization of Fe3O4@IL@MIP Fig. 2(A) illustrates the FT-IR spectra of Fe3O4, chloro-functionalized Fe3O4, Fe3O4@IL, and Fe3O4@IL@MIP nanoparticles. For the bare Fe3O4 nanoparticles, the broad bands at around 645 cm-1 can be attributed to Fe–O vibrations [29]. In the spectrum of chloro-functionalized Fe3O4 nanoparticles, a band at 1080 cm-1 is related to Si– O–Si anti-symmetric stretching vibration, while the bands at 2861 cm-1 and 2924 cm-1 are ascribed to –CH2 stretching vibration [30]. Analysis of both the spectra (Fe3O4 and chloro-functionalized Fe3O4) prove the inarching of 3-cloropropyl trimethoxysilane on Fe3O4 nanoparticles. The spectrum of Fe3O4@IL shows the characteristic bands of imidazolium ring at 1550 cm-1 and 1459 cm-1 [31-32]. After immobilizing BSA followed by PDA coating, a brand new band at 1290 cm-1 appears due to the aromatic ring of PDA [33-34]. Furthermore, the peak at 3400 cm-1 of Fe3O4@IL@MIP nanoparticles exhibits a tendency to become broad, which might be caused by the overlapping peaks of hydroxyls, adsorbed water and amines from PDA [35]. Therefore, these results suggest the successful preparation of Fe3O4@IL@MIP nanoparticles.
Fig. 2. FT-IR spectra (A) and TGA (B) analysis of Fe3O4, chloro-functionalized Fe3O4, Fe3O4@IL and Fe3O4@IL@MIP nanoparticle
To further study the relative composition of Fe3O4@IL@MIP nanoparticles, we performed TGA. Fig. 2(B) shows that the weight loss of chloro-functionalized Fe3O4 is approximately 5.2% when increasing the temperature from 0 ºC to 700 ºC. The amount of weight loss for chloro-functionalized Fe3O4 is higher than that of the bare Fe3O4, indicating the successful grafting of 3-cloropropyl trimethoxysilane. Compared with chloro-functionalized Fe3O4, Fe3O4@IL retained 93.9% of its weight at 700 ºC, suggesting the formation of ionic liquids group via alkylation reaction. Finally, the weight loss of Fe3O4@IL@MIP could reach 14.4%, confirming that PDA had been coated on the surface of Fe3O4@IL through DA self-polymerization process. Fig. 3(A) illustrates the XRD patterns of the prepared nanoparticles. Several diffraction peaks that confirm the Fe3O4 sample exhibiting patterns are in agreement with those of standard Fe3O4 particles. The known standard Fe3O4 diffraction patterns are indexed to (220), (311), (400), (422), (511) and (440) planes of a cubic unit cell, corresponding to the reflection of the inverse spinel structure (JCPDS entry 19-0629). As for Fe3O4@IL and Fe3O4@IL@MIP, the obvious broad peak at 2θ value of 23º can be assigned to amorphous SiO2 [36], indicating the successful grafting of silane-coupling agent. Furthermore, the main diffraction line of Fe3O4@IL@MIP is similar to Fe3O4, indicating that the crystal structure of magnetic nanoparticles do not suffer any changes upon the coating with PDA.
Fig. 3. XRD patterns (A) and magnetization curve (B) of Fe3O4, Fe3O4@IL and Fe3O4@IL@MIP
To investigate the magnetic properties of the prepared nanoparticles, we used VSM. Fig. 3(B) illustrates the magnetic hysteresis loops of the dried samples at 25 ºC. It is obvious that there is no hysteresis for bare Fe3O4 nanoparticles, indicating that the sample is super-paramagnetic. The saturation magnetization value of bare Fe3O4 nanoparticles is 50.53 emu g-1. Compared with Fe3O4, the magnetization values of Fe3O4@IL and Fe3O4@IL@MIP nanoparticles are 39.22 and 30.17 emu g-1, respectively. The decrease in the magnetization value can be attributed to the grafting of silane-coupling agent and imprinted shell, influencing the magnetic response of Fe3O4 nanoparticles. However, the magnetic Fe3O4@IL@MIP nanoparticles still shows a strong magnetic strength, thus allowing effective magnetic separation. As shown in inset of Fig.3 (B), the as-prepared Fe3O4@IL@MIP can be readily dispersed in solution and subsequently separated from the dispersion within three seconds by an external magnetic field. The TEM images presented in Fig. 4 reveal the morphological structures of Fe3O4, chloro-functionalized Fe3O4, Fe3O4@IL as well as Fe3O4@IL@MIP with different polymerization times of DA. The Fe3O4 nanoparticles show relatively regular spherical in shape with the size of about 250 nm (Fig. 4(A)). After successive modification with 3-cloropropyl trimethoxysilane and VIM, the size and shape of chloro-functionalized Fe3O4 and Fe3O4@IL did not change much from those of Fe3O4 (Figs. 4(B) and 4(C)), suggesting that the grafting of 3-chloropropyl trimethoxysilane on Fe3O4 would be thin. Similar phenomenon has also been reported [37]. In addition, Figs. 4(D)~(F) show the TEM images of Fe3O4@IL@MIP nanoparticles with different DA polymerization time, and a well-defined imprinted layer was deposited on the surface of Fe3O4@IL. There is an interface, which could be easily distinguished between the inner Fe3O4@IL core and the PDA layer. We found that the thickness of PDA layer
had increased with increase in reaction time; the thicknesses of those layers are about 10 nm, 20 nm and 30 nm with the respective reaction times of 2 h, 4 h, and 6 h.
Fig. 4. TEM images of Fe3O4 (A), chloro-functionalized Fe3O4 (B), Fe3O4@IL (C), Fe3O4@IL@MIP-2h (D), Fe3O4@IL@MIP-4h (E), and Fe3O4@IL@MIP-6h (F)
Since the control of imprinted layer is important for surface imprinting technology, we investigated the influence of PDA shell thickness on adsorption and recognition ability of Fe3O4@IL@MIP. Fig. 5 shows that the adsorption capacity and IF value of Fe3O4@IL@MIP obviously increase to a maximum and then decrease with shell thickness changing from 10 nm to 30 nm. The dimension of BSA is 14 nm × 4 nm × 4 nm [38]. Therefore, when the thickness of imprinted layer is about 10 nm, it might not construct an integrated shape and structure for BSA, thus influencing the adsorption and recognition ability of Fe3O4@IL@MIP. However, the template protein could not be well eluted as the imprinted layer was too thick (30 nm). We found that 20 nm was an optimum imprinted layer thickness that provided high recognition ability of the imprinted cavities. Hence, we used Fe3O4@IL@MIP with imprinted layer thickness of 20 nm for further adsorption experiments.
Fig. 5. Influence of PDA shell thickness on adsorption and recognition ability of Fe3O4@IL@MIP
3.4 Adsorption properties of Fe3O4@IL@MIP To determine the rate of adsorption and the binding capacities as a function of time, we carried out the adsorption kinetics studies using 1.0 mg mL-1 BSA solution at 25 ºC for Fe3O4@IL@MIP and Fe3O4@IL@NIP nanoparticles. From the Fig. 6(A), it is evident that the binding of BSA on both Fe3O4@IL@MIP and Fe3O4@IL@NIP has a fast adsorption profile up to 40 min, after which it gradually slows down. After 60 min, the adsorption reaches equilibrium. Compared with the previous researches [39-40], we found a higher adsorption rate for Fe3O4@IL@MIP nanoparticles. The relative rapid adsorption rate might be attributed to the nano-sized thickness of the PDA layer formed over the nano-supports [41].
Fig. 6. Adsorption kinetics (A) and isotherm curve (B) of Fe3O4@IL@MIP and Fe3O4@IL@NIP at 25 ºC
To further investigate the adsorption kinetics, we applied the pseudo-first-order kinetic model and pseudo-second-order kinetic model to fit the kinetic data. The pseudo-first-order equation (1) and pseudo-second-order equation (2) are expressed as followed,
ln(Qe Qt ) lnQe k1t
(1)
t 1 t 2 Qt k2Qe Qe
(2)
where Qe (mg g-1) and Qt (mg g-1) are the adsorption capacities at equilibrium and the adsorption amount at time t (min), respectively. k1 (min-1) and k2 (mg g-1 min-1) are pseudo-first-order and pseudo-second-order rate constants of adsorption, respectively. Pseudo-first-order model describes the rate of occupation of the adsorption sites to be proportional to the number of unoccupied sites, whereas pseudo-second-order model assumes the chemical adsorption occurring through sharing or exchange of electrons between the adsorbate and adsorbent [42]. Table 1 presents the parameters of kinetic models. We selected the best-fit model based on linear regression correlation coefficient (R2) and found that the pseudo-second-order model fitted the experimental data better with R2 value higher than 0.98, suggesting that the rate-limiting step might be due to chemical adsorption. Furthermore, the k2 and Qe values of Fe3O4@IL@MIP are much higher than those of Fe3O4@IL@NIP. This indicates that the interactions between Fe3O4@IL@MIP and BSA are much stronger compared with those between Fe3O4@IL@NIP and BSA, probably because of the formation of BSA-sized cavities in the imprinted particles. Table 1. Model and parameters of adsorption kinetics and isotherms of Fe3O4@IL@MIP and Fe3O4@IL@NIP
Model
Parameters
Fe3O4@IL@MIP
Fe3O4@IL@NIP
Pseudo-first-order kinetics
k1 (min-1)
0.0642
0.0449
Qe (mg g-1)
77.4
21.9
R2
0.93
0.91
k2 (g mg-1 min-1)
1.8×10-4
0.7×10-4
Qe (mg g-1)
101.0
66.2
R2
0.99
0.98
Km (mL mg-1)
1.09
0.72
Qm (mg g-1)
99.0
35.5
Pseudo-second-order kinetics
Langmuir
R2
0.99
0.98
In characterizing the adsorption behaviors of the Fe3O4@IL@MIP and Fe3O4@IL@NIP, the nanoparticles were subjected to the isothermal experiments. Fig. 6(B) shows a rapid increase of adsorption capacity of Fe3O4@IL@MIP with increasing BSA concentration from 0.1 mg mL-1 to 0.6 mg mL-1, and a saturation platform is reached over 1.0 mg mL-1. The amounts of BSA adsorbed onto Fe3O4@IL@MIP are much higher than those of Fe3O4@IL@NIP, indicating the specific recognition sites complementary to the template protein with functional group, molecular size and spatial structure exist in the imprinted layer, while the adsorption of Fe3O4@IL@NIP might be dominated by non-specific binding. In order to further investigate the template affinity of Fe3O4@IL@MIP and Fe3O4@IL@NIP, adsorption isotherms are described by the Langmuir model equation (3),
Ce 1 C e Qe KmQm Qm
(3)
where Qm and Km are the Langmuir constants related to the theoretical maximum adsorption capacity and median binding affinity, respectively. As shown in Table 1, linear fitting of the data to the Langmuir equation yields a good fit for both Fe3O4@IL@MIP and Fe3O4@IL@NIP (with the regression coefficient higher than 0.98), indicating a monolayer adsorption behavior. In addition, the estimated Km and Qm value of Fe3O4@IL@MIP is significantly larger compared with Fe3O4@IL@NIP, further indicating the existence of imprinted cavities in Fe3O4@IL@MIP. 3.5 Rebinding specificity of Fe3O4@IL@MIP To investigate the selective recognition ability of Fe3O4@IL@MIP for BSA, we performed a selectivity experiment. Depending on different molecular weights (Mw) and isoelectric point (pI), we selected three types of proteins, OVA (Mw 45 kDa, pI 4.7), Hb (Mw 64.5 kDa, pI 6.8-7.0) and Lyz (Mw 14 kDa, pI 10.7) as the reference proteins. Table 2 presents the adsorption capacities of Fe3O4@IL@MIP and Fe3O4@IL@NIP for different proteins in pH 8.0 buffered solutions at a protein concentration of 1.0 mg mL-1. We found that the adsorption of Lyz on both nanoparticles (MIP and NIP) were significantly larger compared to the other proteins. According to previous researches, PDA modified surface becomes electronegative in the neutral condition [43-45], thus could adsorb Lyz via nonspecific electrostatic interaction. However, the adsorption capacity of Lyz on Fe3O4@IL@MIP were much lower than those on Fe3O4@IL@NIP, which might be due to electrostatic repulsion between Lyz and ionized imidazole group in imprinted cavities. This also explains the lower affinity of Fe3O4@IL@NIP towards OVA and relatively higher IF value of Fe3O4@IL@MIP for OVA when compared with Hb and Lyz. Furthermore, Hb has a
low electric charge at pH 8.0, thus the modest adsorption capacities for both nanoparticles can be expected due to their weak electrostatic interactions. Hence, we obtained the highest selective factor (β = 4.97) for Fe3O4@IL@MIP when subjected to Lyz, and the selectivity of Fe3O4@IL@MIP on OVA and Hb were also appropriated with the respective β value of 2.41 and 3.03. Table 2. Adsorption capacity, imprinting factors and selectivity coefficients of BSA, OVA and Hb for Fe 3O4@IL@MIP and Fe3O4@IL@NIP
BSA
OVA
Hb
Lyz
Q (mg g-1) -Fe3O4@IL@MIP
50.6
19.8
22.3
25.4
Q (mg g-1) -Fe3O4@IL@NIP
15.2
14.3
20.2
37.6
IF
3.33
1.38
1.10
0.67
β
—
2.41
3.03
4.97
A competitive adsorption experiment, carried out in presence of a binary protein mixture of template and its analogue, is more effective to compare the specific recognition ability for BSA between Fe3O4@IL@MIP and Fe3O4@IL@NIP. According to the data presented in Table 3, Fe3O4@IL@NIP shows a certain degree of “selectivity” towards OVA and Hb with α value of 0.88 and 0.42, respectively. This could be due to the larger molecular size and stronger electrostatic repulsion between BSA and PDA, which made BSA molecule harder to be adsorbed on the surface of Fe3O4@IL@NIP. On the contrary, we found good specific recognition ability of Fe3O4@IL@MIP towards BSA due to the existence of imprinted cavities; Fe3O4@IL@MIP was able to isolate BSA from a binary protein mixture 1 and 2 with corresponding α values of 2.53 and 3.07, respectively. In order to further investigate the specific recognition ability of Fe3O4@IL@MIP for BSA, the tolerance concentration studies of each analogue were carried out in competitive adsorption experiment. As seen in Fig.S2 and Fig.S3, the adsorption capacity of Fe3O4@IL@MIP for BSA in the binary protein mixture decreased with the increase of analogue concentration, while the adsorption capacity of Fe3O4@IL@MIP for analogue was obviously increased at the same time. The α value for Fe3O4@IL@MIP was about 1 when the concentrations of OVA and Hb in their binary protein mixtures reached 1.6 mg mL-1 and 1.8 mg mL-1 respectively. It demonstrated that Fe3O4@IL@MIP had lost their specific recognition ability to separate BSA from the binary protein mixture at this concentration of analogues.
Table 3. The separation factors of Fe3O4@IL@MIP and Fe3O4@IL@NIP
Protein mixture 1
Protein mixture 2
BSA
OVA
α
BSA
Hb
α
Q (mg g-1) -Fe3O4@IL@MIP
38.7
15.3
2.53
41.4
13.5
3.07
Q (mg g-1) -Fe3O4@IL@NIP
9.8
11.2
0.88
7.6
18.3
0.42
3.6 Real sample analysis In order to demonstrate the practical applicability of the new imprinted material, we chose bovine calf serum (BCS), a real sample, from which we isolated BSA using Fe3O4@IL@MIP nanoparticles. Fig. 7 illustrates the results of isolation of BSA from many contaminant proteins present in BCS using SDS-PAGE gel-electrophoresis. The multiple transverse lines in lane 2 reveal the presence of other contaminant proteins in BCS besides BSA. In the SDS-PAGE analysis for Fe3O4@IL@NIP, three obvious lines in lane 4 shows extraction of BSA and other two contaminant proteins by Fe3O4@IL@NIP through nonspecific adsorption. However, compared with Fe3O4@IL@NIP, we found only one line in lane 3, much deeper in color at 66.2 kDa representing BSA, for Fe3O4@IL@MIP, exhibiting good specific recognition ability. From the results obtained from SDS-PAGE analysis, we can infer that Fe3O4@IL@MIP nanoparticles possess numerous imprinted cavities complementary to the template protein BSA; hence, this new imprinted material would be useful in isolating BSA from biological samples.
Fig. 7. SDS-PAGE analysis of the results for the separation of BSA from BCS; lane 1, protein molecular weight marker; lane 2, 10-fold dilution of BCS; lane 3, the protein eluted from Fe3O4@IL@MIP; lane 4, the protein eluted from Fe3O4@IL@NIP.
3.7 Reusability of Fe3O4@IL@MIP
Reusability is an important criterion for a material to be applicable in practical purposes. In order to demonstrate the reusability of Fe3O4@IL@MIP, we repeated the adsorption-desorption cycle six times in BSA solution (Tris-HCl buffer, pH 8.0, 10 mM) with a feed concentration of 1.0 mg mL-1. After adsorption, the Fe3O4@IL@MIP was washed repeatedly with 0.1 M NaCl solution to remove the embedded and loosely adhered BSA. Fig. S4 shows that Fe3O4@IL@MIP nanoparticles lose only 9.68% of its adsorption capacity even after six times cycles, whereas the adsorption capacity of Fe3O4@IL@NIP remains unchanged. This could be due to the change in shape and distribution of imprinted cavities in Fe3O4@IL@MIP during washing [46-47]. On the contrary, since there are no imprinted cavities in Fe3O4@IL@NIP, the affinity of NIP towards any protein is nonspecific; consequently, the effect of washing is negligible. Although Fe3O4@IL@MIP loses its adsorption capacity by 9.68% after sixth cycle, it retains a high recognition ability towards the template protein BSA. Based on the above result, we can confirm that the imprinted nanoparticles possess excellent reusability, required for its application in the detection and separation of protein in real samples.
4. Conclusions This work demonstrates the beneficial combination of surface imprinting technology with protein immobilization based on IL functionalized substrate to prepare BSA size-tailored imprinted nanoparticles. Zeta potential study confirmed immobilization of BSA on Fe3O4@IL via multiple interactions. An optimum imprinted layer (PDA layer) thickness of 20 nm resulted in strong recognition ability of Fe3O4@IL@MIP with highest IF value. Fe3O4@IL@MIP nanoparticles not only exhibited fast adsorption rate and high adsorption capacity, they also demonstrated high specificity and selectivity towards the template protein BSA. Furthermore, reusability as well as the ability of Fe3O4@IL@MIP to isolate BSA from real sample such as BCS makes it a strong contender to be applied in protein purification and separation science. We conclude that the strategy to combine surface imprinting method with immobilizing template protein on IL modified magnetic nanoparticles can yield smart imprinted materials with rapid adsorption rate, high adsorption capacity, high specificity and selectivity, thus advancing the field of protein imprinting one step further.
Acknowledgements This Project Supported by the Foundation of Key Laboratory of Pulp and Paper Science and Technology of Ministry of Education/Shandong Province of China (Grant no. KF201627) and the National Nature Science Foundation of China (Grant no. 21174111 and 51433008).
This Work Supported by Northwestern Polytechnical University through using its high performance computing facilities.
References [1] W.J. Cheong, F. Ali, J.H. Choi, J.O. Lee, K. Yune Sung, Recent applications of molecular imprinted polymers for enantio-selective recognition, Talanta. 106 (2013) 45-59. [2] A. Martín-Esteban, Molecularly-imprinted polymers as a versatile, highly selective tool in sample preparation, TrAC Trends in Analytical Chemistry. 45 (2013) 169-181. [3] N. Atar, T. Eren, M.L. Yola, A molecular imprinted SPR biosensor for sensitive determination of citrinin in red yeast rice, Food Chem. 184 (2015) 7-11. [4] T. Eren, N. Atar, M.L. Yola, H. Karimi-Maleh, A sensitive molecularly imprinted polymer based quartz crystal microbalance nanosensor for selective determination of lovastatin in red yeast rice, Food Chem. 185 (2015) 430-436. [5] S. Kunath, M. Panagiotopoulou, J. Maximilien, N. Marchyk, J. Sänger, K. Haupt, Cell and tissue imaging with molecularly imprinted polymers as plastic antibody mimics, Adv Healthc Mater. 4 (2015) 1322-1326. [6] K. Rostamizadeh, M. Vahedpour, S. Bozorgi, Synthesis, characterization and evaluation of computationally designed nanoparticles of molecular imprinted polymers as drug delivery systems, Int J Pharmaceut. 424 (2012) 67-75. [7] D.R. Kryscio, N.A. Peppas, Critical review and perspective of macromolecularly imprinted polymers, Acta Biomater. 8 (2012) 461-473. [8] S. Li, S. Cao, M.J. Whitcombe, S.A. Piletsky, Size matters: Challenges in imprinting macromolecules, Prog Polym Sci. 39 (2014) 145-163. [9] D. Li, Y. Qin, H. Li, X. He, W. Li, Y. Zhang, A “turn-on” fluorescent receptor for detecting tyrosine phosphopeptide using the surface imprinting procedure and the epitope approach, Biosensors and Bioelectronics. 66 (2015) 224-230. [10] Y. Lv, T. Tan, F. Svec, Molecular imprinting of proteins in polymers attached to the surface of nanomaterials for selective recognition of biomacromolecules, Biotechnol Adv. 31 (2013) 1172-1186. [11] L. Qian, X. Hu, P. Guan, D. Wang, J. Li, C. Du, R. Song, C. Wang, W. Song, The effectively specific recognition of bovine serum albumin imprinted silica nanoparticles by utilizing a macromolecularly functional monomer to stabilize and imprint template, Anal Chim Acta. 884 (2015) 97-105. [12] X. Qu, F. Wang, Y. Sun, Y. Tian, R. Chen, X. Ma, C. Liu, Selective extraction of bioactive glycoprotein in neutral environment through Concanavalin a mediated template immobilization and dopamine surface imprinting, RSC Adv. 6 (2016) 86455-86463.
[13] S. Wang, Jin Ye, Z. Bie, Z. Liu, Affinity-tunable specific recognition of glycoproteins via boronate affinity-based controllable oriented surface imprinting, Chem Sci. 5 (2014) 1135-1140. [14] Y. Hao, R. Gao, D. Liu, B. Zhang, Y. Tang, Z. Guo, Preparation of biocompatible molecularly imprinted shell on superparamagnetic iron oxide nanoparticles for selective depletion of bovine hemoglobin in biological sample, J Colloid Interf Sci. 470 (2016) 100-107. [15] R. Gao, Y. Hao, L. Zhang, X. Cui, D. Liu, M. Zhang, Y. Tang, Y. Zheng, A facile method for protein imprinting on directly carboxyl-functionalized magnetic nanoparticles using non-covalent template immobilization strategy, Chem Eng J. 284 (2016) 139-148. [16] M. Sprynskyy, T. Kowalkowski, H. Tutu, E.M. Cukrowska, B. Buszewski, Ionic liquid modified diatomite as a new effective adsorbent for uranium ions removal from aqueous solution, Colloids and Surfaces A: Physicochemical and Engineering Aspects. 465 (2015) 159-167. [17] G. Shen, X. Zhang, Y. Shen, S. Zhang, L. Fang, One-step immobilization of antibodies for α-1-fetoprotein immunosensor based on dialdehyde cellulose/ionic liquid composite, Anal Biochem. 471 (2015) 38-43. [18] C.J. Carrasco, F. Montilla, L. Bobadilla, S. Ivanova, B.A.G. José Antonio Odriozola A, Oxodiperoxomolybdenum complex immobilized onto ionic liquid modified SBA-15 as an effective catalysis for sulfide oxidation to sulfoxides using hydrogen peroxide, Catal Today. 255 (2015) 102-108. [19] C. Du, X. Hu, P. Guan, Xumian Gao, R. Song, J. Li, L. Qian, N. Zhang, L. Guoa, Preparation of surface-imprinted microspheres effectively controlled by orientated template immobilization using highly cross-linked raspberry-like microspheres for the selective recognition of an immunostimulating peptide, J Mater Chem B. 4 (2016) 1510-1519. [20] S. Sasaki, T. Ooya, Y. Kitayama, T. Takeuchi, Molecularly imprinted protein recognition thin films constructed by controlled/living radical polymerization, J Biosci Bioeng. 119 (2015) 200-205. [21] S. Li, K. Yang, J. Liu, B. Jiang, L. Zhang, Y. Zhang, Surface-Imprinted nanoparticles prepared with a His-Tag-Anchored epitope as the template, Anal Chem. 87 (2015) 4617-4620. [22] X. Tang, W. Liu, J. Chen, J. Jia, Z. Ma, Q. Shi, Y. Gao, X. Wang, S. Xu, K. Wang, P. Guo, D. He, Preparation and evaluation of polydopamine imprinting layer coated multi-walled carbon nanotubes for the determination of testosterone in prostate cancer LNcap cells, Anal Methods-UK. 7 (2015) 8326-8334. [23] W. Wu, L. Yang, F. Zhao, B. Zeng, A vanillin electrochemical sensor based on molecularly imprinted poly(1-vinyl-3-octylimidazole hexafluoride phosphorus) − multi-walled carbon nanotubes@polydopamine – carboxyl single-walled carbon nanotubes composite, Sensors and Actuators B: Chemical. 239 (2017) 481-487. [24] G. Wu, J. Li, X. Qu, Y. Zhang, H. Hong, C. Liu, Template size matched film thickness for effectively in situ surface imprinting: A model study of glycoprotein imprints, RSC Advances. 5 (2015) 47010-47021. [25] M. Lin, H. Huang, Z. Liu, Y. Liu, J. Ge, Y. Fang, Growth–dissolution–regrowth transitions of Fe3O4 nanoparticles as building blocks for 3D magnetic nanoparticle clusters under hydrothermal conditions, Langmuir. 29 (2013) 15433-15441.
[26] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, Jr. J. A. Montgomery, J. E Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. R. Cossi, N. J. Millam, M. Knox Klene, J. B.Cross, V. Bakken, C. Adamo, J. Jaramillo, R. E. Stratmann Gomperts, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. Martin, R. L. Morokuma, V. G.Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, D. J. Fox, Gaussian. Inc.: Wallingford, CT, 2013. [27] G. Guan, S. Zhang, S. Liu, Y. Cai, M. Low, C. P. Teng, I. Y. Phang, Y. Cheng, K. L. Duei, B. M. Srinivasan, Y. Zheng, Y. Zhang, M. Han, Protein induces layer-by-layer exfoliation of transition metal dichalcogenides, Journal of the American Chemical Society. 137 (2015) 6152-6155. [28] W. Song, Y. Liu, L. Qian, L. Niu, L. Xiao, Y. Hou, Y. Wang, X. Fan, Hyperbranched polymeric ionic liquid with imidazolium backbones for highly efficient removal of anionic dyes, Chem Eng J. 287 (2016) 482-491. [29] Z. Zhang, F. Zhang, Q. Zhu, W. Zhao, B. Ma, Y. Ding, Magnetically separable polyoxometalate catalyst for the oxidation of dibenzothiophene with H2O2, J Colloid Interf Sci. 360 (2011) 189-194. [30] Y. Jiang, C. Guo, H. Xia, I. Mahmood, C. Liu, H. Liu, Magnetic nanoparticles supported ionic liquids for lipase immobilization: Enzyme activity in catalyzing esterification, Journal of Molecular Catalysis B: Enzymatic. 58 (2009) 103-109. [31] B. Gao, P. Guan, X. Hu, L. Qian, Q. Wang, L. Yang, D. Wang, The performance optimization and specific adsorption of L-phenylalanine imprinted polymers using 1-vinyl-3-carboxymethylimidazolium chloride as functional monomer, Designed Monomer and Polymers. 18 (2015) 185-198. [32] Y. Lin, F. Wang, Z. Zhang, J. Yang, Y. Wei, Polymer-supported ionic liquids: Synthesis, characterization and application in fuel desulfurization, Fuel. 116 (2014) 273-280. [33] S. Zhang, Y. Zhang, G. Bi, J. Liu, Z. Wang, Q. Xu, H. Xu, X. Li, Mussel-inspired polydopamine biopolymer decorated with magnetic nanoparticles for multiple pollutants removal, J Hazard Mater. 270 (2014) 27-34. [34] M. Müller, B. Torger, E. Bittrich, E. Kaul, L. Ionov, P. Uhlmann, M. Stamm, In-situ ATR-FTIR for characterization of thin biorelated polymer films, Thin Solid Films. 556 (2014) 1-8. [35] Y. Wang, S. Wang, H. Niu, Y. Ma, T. Zeng, Y. Cai, Z. Meng, Preparation of polydopamine coated Fe3O4 nanoparticles and their application for enrichment of polycyclic aromatic hydrocarbons from environmental water samples, J Chromatogr a. 1283 (2013) 20-26. [36] Y. Zhang, M. Zhang, J. Yang, L. Ding, J. Zheng, J. Xu, Facile synthesis of sea urchin-like magnetic copper silicate hollow spheres for efficient removal of hemoglobin in human blood, J Alloy Compd. 695 (2017) 3256-3266. [37] H. He, G. Fu, Y. Wang, Z. Chai, Y. Jiang, Z. Chen, Imprinting of protein over silica nanoparticles via surface graft
copolymerization using low monomer concentration, Biosensors and Bioelectronics. 26 (2010) 760-765. [38] A.K. Wright, M.R. Thompson, Hydrodynamic structure of bovine serum albumin determined by transient electric birefringence, Biophys J. 15(2 Pt 1) (1975) 137-141. [39] M. Zhang, Y. Wang, X. Jia, M. He, M. Xu, S. Yang, C. Zhang, The preparation of magnetic molecularly imprinted nanoparticles for the recognition of bovine hemoglobin, Talanta. 120 (2014) 376-385. [40] X. Li, B. Zhang, L. Tian, W. Li, H. Zhang, Q. Zhang, Improvement of recognition specificity of surface protein-imprinted
magnetic
microspheres
by
reducing
nonspecific
adsorption
of
competitors
using
2-methacryloyloxyethyl phosphorylcholine, Sensors and Actuators B: Chemical. 208 (2015) 559-568. [41] L. Qian, X. Hu, P. Guan, D. Wang, J. Li, C. Du, R. Song, C. Wang, W. Song, The effectively specific recognition of bovine serum albumin imprinted silica nanoparticles by utilizing a macromolecularly functional monomer to stabilize and imprint template, Anal Chim Acta. 884 (2015) 97-105. [42] Y. Liu, Z. Liu, J. Gao, J. Dai, J. Han, Y. Wang, J. Xie, Y. Yan, Selective adsorption behavior of Pb(II) by mesoporous silica SBA-15-supported Pb(II)-imprinted polymer based on surface molecularly imprinting technique, J Hazard Mater. 186 (2011) 197-205. [43] J. Fu, Z. Chen, M. Wang, S. Liu, J. Zhang, J. Zhang, R. Han, Q. Xu, Adsorption of methylene blue by a high-efficiency adsorbent (polydopamine microspheres): Kinetics, isotherm, thermodynamics and mechanism analysis, Chem Eng J. 259 (2015) 51-61. [44] C. Ho, S. Ding, The pH-controlled nanoparticles size of polydopamine for anti-cancer drug delivery, J Mater Sci: Mater Med. 24 (2013) 2381-2390. [45] Y. Yu, J.G. Shapter, R. Popelka-Filcoff, J.W. Bennett, A.V. Ellis, Copper removal using bio-inspired polydopamine coated natural zeolites, J Hazard Mater. 273 (2014) 174-182. [46] X. Li, B. Zhang, W. Li, X. Lei, X. Fan, L. Tian, H. Zhang, Q. Zhang, Preparation and characterization of bovine serum albumin surfaceimprinted thermosensitive magnetic polymer microsphere and its application for protein recognition, Biosensors and Bioelectronics. 51 (2014) 261-267. [47] D. G. Riveros, K. Cordova, C. Michiels, H. Verachtert, G. Derdelinckx, Polydopamine imprinted magnetic nanoparticles as a method to purify and detect class II hydrophobins from heterogeneous mixtures, Talanta. 160 (2016) 761-767.
Highlights Template immobilization was combined with surface imprinting technology. Ionic liquid functionalized Fe3O4 nanoparticles was utilized as substrate. Dopamine was chosen to prepare template-size tailored imprinted layers. The imprinted nanoparticle showed high adsorption capacity and excellent selectivity.
Graphical Abstract
PII: DOI: Reference:
S0039-9140(17)30346-6 http://dx.doi.org/10.1016/j.talanta.2017.03.044 TAL17389
To appear in: Talanta Received date: 14 January 2017 Revised date: 5 March 2017 Accepted date: 16 March 2017 Cite this article as: Liwei Qian, Jiexuan Sun, Chen Hou, Jinfan Yang, Yongwei Li, Dan Lei, Miaoxiu Yang and Sufeng Zhang, Immobilization of BSA on ionic liquid functionalized magnetic Fe3O4 nanoparticles for use in surface imprinting strategy, Talanta, http://dx.doi.org/10.1016/j.talanta.2017.03.044 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Immobilization of BSA on ionic liquid functionalized magnetic Fe3O4 nanoparticles for use in surface imprinting strategy Liwei Qiana,b*, Jiexuan Suna, Chen Houa,b, JinfanYanga,b, Yongwei Lia,b, Dan Leia, Miaoxiu Yanga, Sufeng Zhanga, a
College of Bioresources Chemical and Materials Engineering, Shaanxi University of Science and Technology,
Xi’an 710021, China b
Key Laboratory of Pulp and Paper Science & Technology of Ministry of Education/Shandong Province, Qilu
University of Technology, Jinan, 250353, China
[email protected] (L. Qian), [email protected] (S. Zhang)
*Corresponding author. Tel.: +86 13669272714
ABSTRACT Combining template immobilization with surface imprinting technology is an effective strategy to overcome the difficulties associated with macromolecular template removal and to achieve high specific recognition ability. In this work, ionic liquid functionalized Fe3O4 nanoparticles were prepared via a simple two-step modification process and were used as substrate to immobilize bovine serum albumin (BSA). The zeta potential study revealed immobilization of BSA on the nanoparticles through multiple interactions, and the immobilization capacity was about nine times higher compared with that of bare Fe3O4. Subsequently, dopamine was utilized as functional monomer to prepare BSA surface imprinted nanoparticles. Fourier transform infrared spectroscopy, thermo-gravimetric analysis and transmission electron microscopy verified the successful preparation of BSA imprinted nanoparticles with core-shell structure. The influence of imprinted layer thickness on recognition ability of imprinted nanoparticles was investigated, and the results suggested that 20 nm was an optimum thickness to achieve the best recognition ability. The adsorption isotherm studies showed that the imprinted nanoparticles had a significantly higher adsorption capacity and stronger binding affinity than the non-imprinted ones. Furthermore, the selective as well as the competitive adsorption studies revealed higher selectivity and recognition ability of the
imprinted nanoparticles for BSA. Therefore, the proposed strategy is an effective way to obtain protein imprinted polymers with high adsorption capacity and good recognition ability, thus would be beneficial for the further development and application of protein imprinting technology. Key words: Protein imprinted polymers; Ionic liquids modification; Protein immobilization; Magnetic nanoparticles;
1. Introduction Molecularly imprinted polymers (MIPs), a kind of artificial synthetic material with highly selective recognition ability for template molecules, have drawn much attention in the past few years [1-2]. Owing to their good stability, resistance to harsh environments and selective recognition ability, MIPs for small molecular template have been successfully applied in a wide variety of fields including applications in biosensors [3], bioseparation [4], medical diagnostics [5], and drug delivery [6], etc. However, unlike small molecules, imprinting of macromolecules, especially for protein, enzymes, DNA, viruses and even cells poses a great challenge because of their huge size, structural complexity, and conformational flexibility [7-8]. To address these issues, a variety of ingenious imprinted strategies, such as epitope imprinting approach [9], surface imprinting approach [10] and imprinting using macromolecular functional monomer and cross-linker [11], etc., have been proposed. Among these approaches, the method that combines template immobilization with surface imprinting technology has been regarded as a potential strategy to effectively overcome the difficulties in macromolecular removal and rebinding [12-13]. This method allows creating thin-film like polymer structures containing imprinting cavities, located at or close to the surface of imprinted polymers, thereby facilitating the removal of template and enhance the specific recognition ability of MIPs. Hao et al. proposed an effective method to prepare surface imprinted nanoparticles via covalent immobilization of hemoglobin [14]. The obtained MIPs showed good specific recognition ability with an imprinting factor of 3.85, and the rapid adsorption equilibration time of MIPs demonstrated the advantage of surface imprinting. However, covalent immobilization method requires rather harsh conditions for template removal, which might affect imprinting stability. Compared with covalent immobilization, non-covalent attachment of protein to the surface of functionalized substrate is more promising in surface imprinting technology due to moderate elution conditions [15]. Nevertheless, adopting single binding pattern, like hydrogen binding or electrostatic binding, might lead to relatively low immobilization capacity. Therefore, adopting multiple binding pattern in non-covalent approach is a potential way to obtain substrate with high immobilization capacity. Based on the above reasons, substrate functionalized with ionic liquids (IL) group of imidazole type has drawn
worldwide attention due to their multiple interactions [16-18]. Recently, our group has reported a surface imprinted immunostimulating peptide on raspberry-like microsphere via non-covalent immobilization [19], and we have used IL functionalized substrate to immobilize template in the work. We found that due to the multiple functional groups of IL, peptide immobilization capacity reached to 22.4 mg g-1, five times higher than the substrate without IL modification. Therefore, immobilization of template using IL functionalized substrate in surface imprinting technology would facilitate preparation of MIPs with high capacity and selectivity. Controlling the thickness of imprinted layer is another critical factor in surface imprinting technology [20-21]. An overmuch thickness of imprinted layer causes embedding of template, while a too thin imprinted layer may create incomplete template-shape cavities. Both situations can reduce selectivity and recognition ability of MIPs. Thus, it is still a developmental trend in macromolecule imprinting to choose or utilize controllable polymeric monomer or method. Dopamine (DA) can self-polymerize to generate polydopamine (PDA) under weak alkaline solution at 25 ºC. By controlling DA concentration and reaction time during polymerization, an adjustable, biocompatible and hydrophilic imprinting layer can easily be obtained [22-23]. Wu et al. reported a series of thickness-tunable imprinting films in nanometer-scale via controlling polymerization time of DA [24] and found that the PDA imprinting film with an optimal thickness exhibited significant selectivity towards glycoprotein, indicating the importance of a template-size matched imprinted layer in MIPs. In order to obtain the MIPs with high adsorption properties and recognition ability, the surface imprinting technology with thickness control is combined with an IL-based template immobilization technology for the first time in this work. First of all, we prepared an IL functionalized Fe3O4 nanoparticle (Fe3O4@IL) via a simple two-step modification process, and utilized Fe3O4@IL nanoparticles to immobilize bovine serum albumin (BSA), which was subsequently imprinted via using DA as a functional monomer. The resulting magnetic nano-materials were characterized by transmission electron microscope (TEM), X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, and vibrating sample magnetometer (VSM). Meanwhile, the adsorption behavior of MIPs was investigated by isothermal rebinding and dynamic adsorption. Furthermore, specific recognition ability of MIPs was investigated using selective adsorption experiment, competitive adsorption experiment, and real sample test.
2. Material and methods 2.1. Materials Dopamine (DA), ferric chloride hexahydrate (FeCl3·6H2O), anhydrous sodium acetate (NaOAc), ethylene
glycol (EG), sodium citrate, bovine serum albumin (BSA), ovalbumin (OVA) and lysozyme (Lyz) were purchased from sigma (St. Louis, MO, USA). (3-Chloropropyl)trimethoxysilan (Cptms) and 1-Methylimidazole were obtained from was supplies by Acros Organics (Morris Plains, NJ, USA). All reagents were of at least analytical grade and used without further treatment. 2.2. Instrumentation Fourier transforms infrared (FTIR) spectra were acquired on a TENSOR27 FTIR spectrometer (Bruker). The samples were prepared by mixing the products with KBr and pressing into a compact pellet. Morphology and structure of the nanoparticles were observed using transmission electron microscopy (TEM, JEOL JEM-3010). The polymer content of these nanoparticles was determined by thermo-gravimetric analysis (TGA, Q50, TA instruments) in the temperature range of 100 to 700 oC, with a heating rate of 10 oC min-1 under nitrogen atmosphere. Room temperature magnetization isotherms were obtained using a vibrating sample magnetometer (VSM, LDJ9600). Zeta potential is measured by using a commercial laser light scattering spectrometer (Zetasizer Nano-ZS). The absorption spectra were measured using a UV spectrophotometer (Varian, Cary-1E). The crystal phase was collected by a PAN analytical X’pert Pro diffractometer (XRD, Almelo). Protein identification was performed using a Shimadzu LC-2010A series HPLC with an Xtimate SEC column (250 mm × 7.8 mm, 5 μm, 300 Å). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a DYY-6C electrophoresis system (Beijing Liuyi instrument plant). 2.3. Synthesis of Fe3O4 nanoparticles Fe3O4 nanoparticles were prepared using a previously reported solvothermal method with some modifications [25]. Briefly, FeCl3·6H2O (3.0 g) was taken in ethylene glycol (100 mL) containing NaOAc (4.8 g) and sodium citrate (0.7 g), and the mixture was heated with vigorous stirring to obtain a homogeneous solution. The solution was then transferred into a 200 mL Teflon reactor and heated at 200 ºC for 10 h, followed by cooling down to 25 ºC. The product was washed several times with deionized water and ethanol and finally, dried in a vacuum at 30 ºC overnight to obtain Fe3O4 nanoparticles. 2.4. Synthesis of chloro-functionalized Fe3O4 nanoparticles A mixture of Fe3O4 nanoparticles (1 g) in dry toluene (25 mL) was sonicated for 30 min, and 3-Cloropropyl trimethoxysilane (0.5 mL) was then added to the dispersed Fe3O4 nanoparticle in toluene. The reaction mixture was slowly heated to 115 ºC and then stirred at this temperature for 24 h. The obtained product was separated by an external magnet and washed three times with diethyl ether and CH2Cl2, followed by drying under vacuum. 2.5. Synthesis of Fe3O4@IL nanoparticles
1-Methylimidazole (0.2 mL) was added dropwise to chloro-functionalized Fe3O4 nanoparticles (0.5 g) in 250 mL ethanol at 60 ºC under a nitrogen atmosphere. Once the addition was complete (30 min), the mixture was stirred for 24 h. The product was separated by an external magnet, washed with ethanol, and finally, dried at 25 ºC in vacuum to obtain Fe3O4@IL nanoparticles. 2.6. Synthesis of Fe3O4@IL@MIP To prepare BSA surface imprinted magnetic nanoparticles, Fe3O4@IL nanoparticles (0.2 g) was first incubated in BSA Tris-HCl buffer (pH 8.0, 10 mM) solution at a concentration of 1.2 mg mL-1. After 3 h incubation, Fe3O4@IL nanoparticles were magnetically separated and washed with deionized water. Subsequently, Fe3O4@IL nanoparticles were dispersed in 100 mL of Tris-HCl buffer (pH 8.0, 10 mM) in a three-necked flask, and DA (0.1 g) was added to the solution with continuous mechanical stirring at 25 ºC for 4 h. The obtained product (designed Fe3O4@IL@MIP) was washed with NaCl solution (0.1 M) to remove the template protein until no adsorption was detected by UV-vis spectrophotometer at about 278 nm. The product was further washed with deionized water to remove NaCl and dried under vacuum. The non-imprinted polymers (designed Fe3O4@IL@NIP) were prepared using the same protocol as Fe3O4@IL@MIP but without the addition of BSA. 2.7. Adsorption experiments of Fe3O4@IL@MIP and Fe3O4@IL@NIP To investigate the adsorption kinetics of the adsorbent, 50 mg of Fe3O4@IL@MIP were suspended in 10 mL of BSA Tris-HCl buffer (pH 8.0, 10 mM) solution at a concentration of 1.0 mg mL-1. The mixture was incubated at regular time intervals from 5 min to 80 min at 25 ºC, and the polymers were magnetically separated from the solution. The concentration of BSA in residual solution was measured by UV-vis spectrophotometer. The binding tests were performed at room temperature (25 ºC) using BSA solutions of different initial concentrations, ranging from 0.1 to 1.2 mg mL-1. After 12 h incubation, which was sufficient to reach adsorption equilibrium, the polymers were magnetically separated from the solution, and the concentrations of solutions were measured by a UV-vis spectrophotometer at 278 nm. The adsorption capacities (Q) of the MIPs for the template protein BSA were calculated using the following formula: Q = (C0 - Ce)V/m, where C0 is the initial concentration of BSA (mg mL-1), Ce is the BSA concentration (mg mL-1) of the supernatant solution, V is the volume of the BSA solution (mL), and m is the mass of MIPs (g). To evaluate the specificity of MIPs toward the BSA template or template analogues, imprinting factor (IF) was used and calculated from the following equation: IF = QMIPs/QNIPs, where QMIPs and QNIPs are the adsorption capacity of the template or template analogues by MIPs and NIPs, respectively. To investigate the selectivity of the imprinted material, 50 mg of Fe3O4@IL@MIP nanoparticles were added to
a centrifuge tube containing 10 mL protein solution (template and template analogue separately) of concentration 1.0 mg mL-1 at 25 ºC and shaken for 12 h. After magnetic separation of Fe3O4@IL@MIP from the solution, the concentration of protein in the supernatant solution was measured using a UV spectrophotometer. The selectivity factor (β) is defined as the following equation: β = IFtemp/ IFana, where IFtemp and IFana are the imprinting factors for the template molecule and for the analogues, respectively. In the competitive adsorption experiments, 50 mg of Fe3O4@IL@MIP nanoparticles were conditioned in a 10 mL binary mixture solution (containing both the template and its analogue) at 25 ºC with each protein concentration of 1.0 mg mL-1. After 12 h incubation, Fe3O4@IL@MIP was magnetically separated from the solution, and the concentrations of template protein and analogue in the supernatant solution were measured using HPLC. The separation factor (α) was calculated to evaluate the separation ability of adsorbing material in a mixture of BSA and its analogues. The separation factor is defined by the following equation: α = QBSA/Qana, where QBSA and Qana are the binding capacities of BSA and its analogues, respectively. In order to further investigate the specific recognition ability of Fe3O4@IL@MIP, the tolerance concentration of BSA’s analogue in the competitive adsorption experiments was carried out. In this experiment, the concentration of BSA was fixed at 1.0 mg mL-1, while the concentration of its analogue was increased from 1.0 mg mL-1 until the α value was about 1. Other conditions were same as above competitive adsorption experiments. In the real sample test, bovine calf serum (BCS) was 10-fold diluted with phosphate buffer (10 mM, pH 8.0). About 50 mg of Fe3O4@IL@MIP was conditioned in the above serum solution at 25 ºC for 12 h. The particles were treated with phosphate buffer (10 mM, pH 8.0) containing 5 mM NaCl to remove the weakly adsorbed proteins and then with 0.1 M NaCl solution to elute the strongly adsorbed proteins. The elution process was continued until no characteristic peaks of these proteins showed in the UV region of the spectrum. The eluate for strongly adsorbed proteins was combined, desalted by using dialysis tubing with a molecular cut-off of 500, and then lyophilized. The obtained products were dissolved using phosphate buffer (10 mM, pH 8.0), and their concentrations were maintained at 1 mg mL-1. To separate and analyze the proteins, gel-electrophoresis was performed using SDS-PAGE (12% polyacrylamide separating gel and 5% polyacrylamide stacking gel); 20 μL of product solution was taken for running SDS-PAGE electrophoresis. 2.8. Computer simulation In order to study the immobilization mechanism of Fe3O4@IL, first-principles calculations based on density functional theory (DFT) were performed to investigate their hydrogen bond and π-π stack interaction (b3lyp/6-31g(d) was used as basis set) [26]. According to the previous report [27], we simplified the models of
Fe3O4@IL and BSA in order to facilitate the computer simulation. Among them, Fe3O4@IL could be simplified as 1-propyl-3-methylimidazole, while the functional groups in BSA could be replaced by CH3-X (X=SH, OH, SSCH3, COOH, NH2, C6H5). The binding energies (ΔE) of the complexes formed from 1-propyl-3-methylimidazole and functional groups in BSA were evaluated by the following equation:
ΔE E (complex) E ( IL) E ( FG) where E(IL) , E(FG) and E(complex) were the potential energies of 1-propyl-3-methylimidazole, functional groups in BSA, and their complex respectively.
3. Results and discussion 3.1 Production process of Fe3O4@IL@MIP Scheme 1 illustrates the synthesis of Fe3O4@IL@MIP, combining the surface imprinting technique and immobilized template strategy. Fe3O4 nanoparticles were first prepared according to a modified solvothermal method and subsequently, the nanoparticles were allowed to react with (3-chloropropyl)trimethoxysilane to obtain chloro-functionalized Fe3O4. In the next step, Fe3O4@IL with abundantly ionized imidazole groups were prepared via alkylation reaction, and BSA was easily immobilized through hydrogen bond, electrostatic, and π-π force interactions. Immobilization of template protein on supporter through non-covalent interactions not only overcomes the disadvantage of the harsh desorption condition for covalent template immobilization, but also possesses the advantage of better imprinting effect to some degree when compared with that achieved from the traditional free-template polymerization process. Finally, an appropriate thickness of PDA layer was coated on the surface of Fe3O4@IL-BSA complex, and Fe3O4@IL@MIP nanoparticles with recognition sites complementary to BSA were obtained after the removal of the embedded template protein by simple elution with NaCl solution.
Scheme 1. Schematic illustration of the synthesis Fe3O4@IL@MIP nanoparticles
3.2 Immobilization of BSA on Fe3O4@IL Since the immobilization process of the template protein BSA on Fe3O4@IL was crucial, their interaction mechanism should be investigated. In this work, we utilized Zeta potential to qualitatively analyze the multiple-interaction between Fe3O4@IL and BSA; meanwhile, we also investigated the influence of BSA concentration on immobilization capacity of Fe3O4@IL. Fig.1 shows that the Zeta potential value of Fe3O4@IL is 26.5 mV at pH 8.0 due to the presence of ionized imidazole group on the surface of Fe3O4@IL. After interacting with BSA (isoelectric point 4.7), Zeta potential value of Fe3O4@IL sharply decreases. The immobilization effect in this process might be dominated by electrostatic interaction. When BSA concentration is 0.4 mg mL-1, the exterior of the Fe3O4@IL appears nearly neutral. Further increasing the concentration of BSA, they could be still adsorbed on Fe3O4@IL, which should be due to hydrogen bond and π-π interaction, resulting in further decrease of Zeta potential value (Hydrogen bond and π-π interaction between Fe3O4@IL and BSA was further confirmed by computer simulation, and results were shown in Table S1 and Fig.S1). We also observed similar phenomenon in our previous research [28]. Finally, the Zeta potential value of Fe3O4@IL is fixed to -15.8 mV at the BSA concentration of 1.2 mg mL-1, when the maximal adsorption capacity is 36.8 mg g-1. This maximal adsorption capacity is significantly higher compared with that of the bare Fe3O4 (3.8 mg g-1) and chloro-functionalized Fe3O4 (5.1 mg g-1) nanoparticles at the same condition. Therefore, the above result demonstrates that Fe3O4@IL could preferably immobilize BSA via multiple interactions.
Fig. 1. Influence of BSA concentration on the Zeta potential and immobilization capacity of Fe3O4@IL at pH 8.0
3.3 Characterization of Fe3O4@IL@MIP Fig. 2(A) illustrates the FT-IR spectra of Fe3O4, chloro-functionalized Fe3O4, Fe3O4@IL, and Fe3O4@IL@MIP nanoparticles. For the bare Fe3O4 nanoparticles, the broad bands at around 645 cm-1 can be attributed to Fe–O vibrations [29]. In the spectrum of chloro-functionalized Fe3O4 nanoparticles, a band at 1080 cm-1 is related to Si– O–Si anti-symmetric stretching vibration, while the bands at 2861 cm-1 and 2924 cm-1 are ascribed to –CH2 stretching vibration [30]. Analysis of both the spectra (Fe3O4 and chloro-functionalized Fe3O4) prove the inarching of 3-cloropropyl trimethoxysilane on Fe3O4 nanoparticles. The spectrum of Fe3O4@IL shows the characteristic bands of imidazolium ring at 1550 cm-1 and 1459 cm-1 [31-32]. After immobilizing BSA followed by PDA coating, a brand new band at 1290 cm-1 appears due to the aromatic ring of PDA [33-34]. Furthermore, the peak at 3400 cm-1 of Fe3O4@IL@MIP nanoparticles exhibits a tendency to become broad, which might be caused by the overlapping peaks of hydroxyls, adsorbed water and amines from PDA [35]. Therefore, these results suggest the successful preparation of Fe3O4@IL@MIP nanoparticles.
Fig. 2. FT-IR spectra (A) and TGA (B) analysis of Fe3O4, chloro-functionalized Fe3O4, Fe3O4@IL and Fe3O4@IL@MIP nanoparticle
To further study the relative composition of Fe3O4@IL@MIP nanoparticles, we performed TGA. Fig. 2(B) shows that the weight loss of chloro-functionalized Fe3O4 is approximately 5.2% when increasing the temperature from 0 ºC to 700 ºC. The amount of weight loss for chloro-functionalized Fe3O4 is higher than that of the bare Fe3O4, indicating the successful grafting of 3-cloropropyl trimethoxysilane. Compared with chloro-functionalized Fe3O4, Fe3O4@IL retained 93.9% of its weight at 700 ºC, suggesting the formation of ionic liquids group via alkylation reaction. Finally, the weight loss of Fe3O4@IL@MIP could reach 14.4%, confirming that PDA had been coated on the surface of Fe3O4@IL through DA self-polymerization process. Fig. 3(A) illustrates the XRD patterns of the prepared nanoparticles. Several diffraction peaks that confirm the Fe3O4 sample exhibiting patterns are in agreement with those of standard Fe3O4 particles. The known standard Fe3O4 diffraction patterns are indexed to (220), (311), (400), (422), (511) and (440) planes of a cubic unit cell, corresponding to the reflection of the inverse spinel structure (JCPDS entry 19-0629). As for Fe3O4@IL and Fe3O4@IL@MIP, the obvious broad peak at 2θ value of 23º can be assigned to amorphous SiO2 [36], indicating the successful grafting of silane-coupling agent. Furthermore, the main diffraction line of Fe3O4@IL@MIP is similar to Fe3O4, indicating that the crystal structure of magnetic nanoparticles do not suffer any changes upon the coating with PDA.
Fig. 3. XRD patterns (A) and magnetization curve (B) of Fe3O4, Fe3O4@IL and Fe3O4@IL@MIP
To investigate the magnetic properties of the prepared nanoparticles, we used VSM. Fig. 3(B) illustrates the magnetic hysteresis loops of the dried samples at 25 ºC. It is obvious that there is no hysteresis for bare Fe3O4 nanoparticles, indicating that the sample is super-paramagnetic. The saturation magnetization value of bare Fe3O4 nanoparticles is 50.53 emu g-1. Compared with Fe3O4, the magnetization values of Fe3O4@IL and Fe3O4@IL@MIP nanoparticles are 39.22 and 30.17 emu g-1, respectively. The decrease in the magnetization value can be attributed to the grafting of silane-coupling agent and imprinted shell, influencing the magnetic response of Fe3O4 nanoparticles. However, the magnetic Fe3O4@IL@MIP nanoparticles still shows a strong magnetic strength, thus allowing effective magnetic separation. As shown in inset of Fig.3 (B), the as-prepared Fe3O4@IL@MIP can be readily dispersed in solution and subsequently separated from the dispersion within three seconds by an external magnetic field. The TEM images presented in Fig. 4 reveal the morphological structures of Fe3O4, chloro-functionalized Fe3O4, Fe3O4@IL as well as Fe3O4@IL@MIP with different polymerization times of DA. The Fe3O4 nanoparticles show relatively regular spherical in shape with the size of about 250 nm (Fig. 4(A)). After successive modification with 3-cloropropyl trimethoxysilane and VIM, the size and shape of chloro-functionalized Fe3O4 and Fe3O4@IL did not change much from those of Fe3O4 (Figs. 4(B) and 4(C)), suggesting that the grafting of 3-chloropropyl trimethoxysilane on Fe3O4 would be thin. Similar phenomenon has also been reported [37]. In addition, Figs. 4(D)~(F) show the TEM images of Fe3O4@IL@MIP nanoparticles with different DA polymerization time, and a well-defined imprinted layer was deposited on the surface of Fe3O4@IL. There is an interface, which could be easily distinguished between the inner Fe3O4@IL core and the PDA layer. We found that the thickness of PDA layer
had increased with increase in reaction time; the thicknesses of those layers are about 10 nm, 20 nm and 30 nm with the respective reaction times of 2 h, 4 h, and 6 h.
Fig. 4. TEM images of Fe3O4 (A), chloro-functionalized Fe3O4 (B), Fe3O4@IL (C), Fe3O4@IL@MIP-2h (D), Fe3O4@IL@MIP-4h (E), and Fe3O4@IL@MIP-6h (F)
Since the control of imprinted layer is important for surface imprinting technology, we investigated the influence of PDA shell thickness on adsorption and recognition ability of Fe3O4@IL@MIP. Fig. 5 shows that the adsorption capacity and IF value of Fe3O4@IL@MIP obviously increase to a maximum and then decrease with shell thickness changing from 10 nm to 30 nm. The dimension of BSA is 14 nm × 4 nm × 4 nm [38]. Therefore, when the thickness of imprinted layer is about 10 nm, it might not construct an integrated shape and structure for BSA, thus influencing the adsorption and recognition ability of Fe3O4@IL@MIP. However, the template protein could not be well eluted as the imprinted layer was too thick (30 nm). We found that 20 nm was an optimum imprinted layer thickness that provided high recognition ability of the imprinted cavities. Hence, we used Fe3O4@IL@MIP with imprinted layer thickness of 20 nm for further adsorption experiments.
Fig. 5. Influence of PDA shell thickness on adsorption and recognition ability of Fe3O4@IL@MIP
3.4 Adsorption properties of Fe3O4@IL@MIP To determine the rate of adsorption and the binding capacities as a function of time, we carried out the adsorption kinetics studies using 1.0 mg mL-1 BSA solution at 25 ºC for Fe3O4@IL@MIP and Fe3O4@IL@NIP nanoparticles. From the Fig. 6(A), it is evident that the binding of BSA on both Fe3O4@IL@MIP and Fe3O4@IL@NIP has a fast adsorption profile up to 40 min, after which it gradually slows down. After 60 min, the adsorption reaches equilibrium. Compared with the previous researches [39-40], we found a higher adsorption rate for Fe3O4@IL@MIP nanoparticles. The relative rapid adsorption rate might be attributed to the nano-sized thickness of the PDA layer formed over the nano-supports [41].
Fig. 6. Adsorption kinetics (A) and isotherm curve (B) of Fe3O4@IL@MIP and Fe3O4@IL@NIP at 25 ºC
To further investigate the adsorption kinetics, we applied the pseudo-first-order kinetic model and pseudo-second-order kinetic model to fit the kinetic data. The pseudo-first-order equation (1) and pseudo-second-order equation (2) are expressed as followed,
ln(Qe Qt ) lnQe k1t
(1)
t 1 t 2 Qt k2Qe Qe
(2)
where Qe (mg g-1) and Qt (mg g-1) are the adsorption capacities at equilibrium and the adsorption amount at time t (min), respectively. k1 (min-1) and k2 (mg g-1 min-1) are pseudo-first-order and pseudo-second-order rate constants of adsorption, respectively. Pseudo-first-order model describes the rate of occupation of the adsorption sites to be proportional to the number of unoccupied sites, whereas pseudo-second-order model assumes the chemical adsorption occurring through sharing or exchange of electrons between the adsorbate and adsorbent [42]. Table 1 presents the parameters of kinetic models. We selected the best-fit model based on linear regression correlation coefficient (R2) and found that the pseudo-second-order model fitted the experimental data better with R2 value higher than 0.98, suggesting that the rate-limiting step might be due to chemical adsorption. Furthermore, the k2 and Qe values of Fe3O4@IL@MIP are much higher than those of Fe3O4@IL@NIP. This indicates that the interactions between Fe3O4@IL@MIP and BSA are much stronger compared with those between Fe3O4@IL@NIP and BSA, probably because of the formation of BSA-sized cavities in the imprinted particles. Table 1. Model and parameters of adsorption kinetics and isotherms of Fe3O4@IL@MIP and Fe3O4@IL@NIP
Model
Parameters
Fe3O4@IL@MIP
Fe3O4@IL@NIP
Pseudo-first-order kinetics
k1 (min-1)
0.0642
0.0449
Qe (mg g-1)
77.4
21.9
R2
0.93
0.91
k2 (g mg-1 min-1)
1.8×10-4
0.7×10-4
Qe (mg g-1)
101.0
66.2
R2
0.99
0.98
Km (mL mg-1)
1.09
0.72
Qm (mg g-1)
99.0
35.5
Pseudo-second-order kinetics
Langmuir
R2
0.99
0.98
In characterizing the adsorption behaviors of the Fe3O4@IL@MIP and Fe3O4@IL@NIP, the nanoparticles were subjected to the isothermal experiments. Fig. 6(B) shows a rapid increase of adsorption capacity of Fe3O4@IL@MIP with increasing BSA concentration from 0.1 mg mL-1 to 0.6 mg mL-1, and a saturation platform is reached over 1.0 mg mL-1. The amounts of BSA adsorbed onto Fe3O4@IL@MIP are much higher than those of Fe3O4@IL@NIP, indicating the specific recognition sites complementary to the template protein with functional group, molecular size and spatial structure exist in the imprinted layer, while the adsorption of Fe3O4@IL@NIP might be dominated by non-specific binding. In order to further investigate the template affinity of Fe3O4@IL@MIP and Fe3O4@IL@NIP, adsorption isotherms are described by the Langmuir model equation (3),
Ce 1 C e Qe KmQm Qm
(3)
where Qm and Km are the Langmuir constants related to the theoretical maximum adsorption capacity and median binding affinity, respectively. As shown in Table 1, linear fitting of the data to the Langmuir equation yields a good fit for both Fe3O4@IL@MIP and Fe3O4@IL@NIP (with the regression coefficient higher than 0.98), indicating a monolayer adsorption behavior. In addition, the estimated Km and Qm value of Fe3O4@IL@MIP is significantly larger compared with Fe3O4@IL@NIP, further indicating the existence of imprinted cavities in Fe3O4@IL@MIP. 3.5 Rebinding specificity of Fe3O4@IL@MIP To investigate the selective recognition ability of Fe3O4@IL@MIP for BSA, we performed a selectivity experiment. Depending on different molecular weights (Mw) and isoelectric point (pI), we selected three types of proteins, OVA (Mw 45 kDa, pI 4.7), Hb (Mw 64.5 kDa, pI 6.8-7.0) and Lyz (Mw 14 kDa, pI 10.7) as the reference proteins. Table 2 presents the adsorption capacities of Fe3O4@IL@MIP and Fe3O4@IL@NIP for different proteins in pH 8.0 buffered solutions at a protein concentration of 1.0 mg mL-1. We found that the adsorption of Lyz on both nanoparticles (MIP and NIP) were significantly larger compared to the other proteins. According to previous researches, PDA modified surface becomes electronegative in the neutral condition [43-45], thus could adsorb Lyz via nonspecific electrostatic interaction. However, the adsorption capacity of Lyz on Fe3O4@IL@MIP were much lower than those on Fe3O4@IL@NIP, which might be due to electrostatic repulsion between Lyz and ionized imidazole group in imprinted cavities. This also explains the lower affinity of Fe3O4@IL@NIP towards OVA and relatively higher IF value of Fe3O4@IL@MIP for OVA when compared with Hb and Lyz. Furthermore, Hb has a
low electric charge at pH 8.0, thus the modest adsorption capacities for both nanoparticles can be expected due to their weak electrostatic interactions. Hence, we obtained the highest selective factor (β = 4.97) for Fe3O4@IL@MIP when subjected to Lyz, and the selectivity of Fe3O4@IL@MIP on OVA and Hb were also appropriated with the respective β value of 2.41 and 3.03. Table 2. Adsorption capacity, imprinting factors and selectivity coefficients of BSA, OVA and Hb for Fe 3O4@IL@MIP and Fe3O4@IL@NIP
BSA
OVA
Hb
Lyz
Q (mg g-1) -Fe3O4@IL@MIP
50.6
19.8
22.3
25.4
Q (mg g-1) -Fe3O4@IL@NIP
15.2
14.3
20.2
37.6
IF
3.33
1.38
1.10
0.67
β
—
2.41
3.03
4.97
A competitive adsorption experiment, carried out in presence of a binary protein mixture of template and its analogue, is more effective to compare the specific recognition ability for BSA between Fe3O4@IL@MIP and Fe3O4@IL@NIP. According to the data presented in Table 3, Fe3O4@IL@NIP shows a certain degree of “selectivity” towards OVA and Hb with α value of 0.88 and 0.42, respectively. This could be due to the larger molecular size and stronger electrostatic repulsion between BSA and PDA, which made BSA molecule harder to be adsorbed on the surface of Fe3O4@IL@NIP. On the contrary, we found good specific recognition ability of Fe3O4@IL@MIP towards BSA due to the existence of imprinted cavities; Fe3O4@IL@MIP was able to isolate BSA from a binary protein mixture 1 and 2 with corresponding α values of 2.53 and 3.07, respectively. In order to further investigate the specific recognition ability of Fe3O4@IL@MIP for BSA, the tolerance concentration studies of each analogue were carried out in competitive adsorption experiment. As seen in Fig.S2 and Fig.S3, the adsorption capacity of Fe3O4@IL@MIP for BSA in the binary protein mixture decreased with the increase of analogue concentration, while the adsorption capacity of Fe3O4@IL@MIP for analogue was obviously increased at the same time. The α value for Fe3O4@IL@MIP was about 1 when the concentrations of OVA and Hb in their binary protein mixtures reached 1.6 mg mL-1 and 1.8 mg mL-1 respectively. It demonstrated that Fe3O4@IL@MIP had lost their specific recognition ability to separate BSA from the binary protein mixture at this concentration of analogues.
Table 3. The separation factors of Fe3O4@IL@MIP and Fe3O4@IL@NIP
Protein mixture 1
Protein mixture 2
BSA
OVA
α
BSA
Hb
α
Q (mg g-1) -Fe3O4@IL@MIP
38.7
15.3
2.53
41.4
13.5
3.07
Q (mg g-1) -Fe3O4@IL@NIP
9.8
11.2
0.88
7.6
18.3
0.42
3.6 Real sample analysis In order to demonstrate the practical applicability of the new imprinted material, we chose bovine calf serum (BCS), a real sample, from which we isolated BSA using Fe3O4@IL@MIP nanoparticles. Fig. 7 illustrates the results of isolation of BSA from many contaminant proteins present in BCS using SDS-PAGE gel-electrophoresis. The multiple transverse lines in lane 2 reveal the presence of other contaminant proteins in BCS besides BSA. In the SDS-PAGE analysis for Fe3O4@IL@NIP, three obvious lines in lane 4 shows extraction of BSA and other two contaminant proteins by Fe3O4@IL@NIP through nonspecific adsorption. However, compared with Fe3O4@IL@NIP, we found only one line in lane 3, much deeper in color at 66.2 kDa representing BSA, for Fe3O4@IL@MIP, exhibiting good specific recognition ability. From the results obtained from SDS-PAGE analysis, we can infer that Fe3O4@IL@MIP nanoparticles possess numerous imprinted cavities complementary to the template protein BSA; hence, this new imprinted material would be useful in isolating BSA from biological samples.
Fig. 7. SDS-PAGE analysis of the results for the separation of BSA from BCS; lane 1, protein molecular weight marker; lane 2, 10-fold dilution of BCS; lane 3, the protein eluted from Fe3O4@IL@MIP; lane 4, the protein eluted from Fe3O4@IL@NIP.
3.7 Reusability of Fe3O4@IL@MIP
Reusability is an important criterion for a material to be applicable in practical purposes. In order to demonstrate the reusability of Fe3O4@IL@MIP, we repeated the adsorption-desorption cycle six times in BSA solution (Tris-HCl buffer, pH 8.0, 10 mM) with a feed concentration of 1.0 mg mL-1. After adsorption, the Fe3O4@IL@MIP was washed repeatedly with 0.1 M NaCl solution to remove the embedded and loosely adhered BSA. Fig. S4 shows that Fe3O4@IL@MIP nanoparticles lose only 9.68% of its adsorption capacity even after six times cycles, whereas the adsorption capacity of Fe3O4@IL@NIP remains unchanged. This could be due to the change in shape and distribution of imprinted cavities in Fe3O4@IL@MIP during washing [46-47]. On the contrary, since there are no imprinted cavities in Fe3O4@IL@NIP, the affinity of NIP towards any protein is nonspecific; consequently, the effect of washing is negligible. Although Fe3O4@IL@MIP loses its adsorption capacity by 9.68% after sixth cycle, it retains a high recognition ability towards the template protein BSA. Based on the above result, we can confirm that the imprinted nanoparticles possess excellent reusability, required for its application in the detection and separation of protein in real samples.
4. Conclusions This work demonstrates the beneficial combination of surface imprinting technology with protein immobilization based on IL functionalized substrate to prepare BSA size-tailored imprinted nanoparticles. Zeta potential study confirmed immobilization of BSA on Fe3O4@IL via multiple interactions. An optimum imprinted layer (PDA layer) thickness of 20 nm resulted in strong recognition ability of Fe3O4@IL@MIP with highest IF value. Fe3O4@IL@MIP nanoparticles not only exhibited fast adsorption rate and high adsorption capacity, they also demonstrated high specificity and selectivity towards the template protein BSA. Furthermore, reusability as well as the ability of Fe3O4@IL@MIP to isolate BSA from real sample such as BCS makes it a strong contender to be applied in protein purification and separation science. We conclude that the strategy to combine surface imprinting method with immobilizing template protein on IL modified magnetic nanoparticles can yield smart imprinted materials with rapid adsorption rate, high adsorption capacity, high specificity and selectivity, thus advancing the field of protein imprinting one step further.
Acknowledgements This Project Supported by the Foundation of Key Laboratory of Pulp and Paper Science and Technology of Ministry of Education/Shandong Province of China (Grant no. KF201627) and the National Nature Science Foundation of China (Grant no. 21174111 and 51433008).
This Work Supported by Northwestern Polytechnical University through using its high performance computing facilities.
References [1] W.J. Cheong, F. Ali, J.H. Choi, J.O. Lee, K. Yune Sung, Recent applications of molecular imprinted polymers for enantio-selective recognition, Talanta. 106 (2013) 45-59. [2] A. Martín-Esteban, Molecularly-imprinted polymers as a versatile, highly selective tool in sample preparation, TrAC Trends in Analytical Chemistry. 45 (2013) 169-181. [3] N. Atar, T. Eren, M.L. Yola, A molecular imprinted SPR biosensor for sensitive determination of citrinin in red yeast rice, Food Chem. 184 (2015) 7-11. [4] T. Eren, N. Atar, M.L. Yola, H. Karimi-Maleh, A sensitive molecularly imprinted polymer based quartz crystal microbalance nanosensor for selective determination of lovastatin in red yeast rice, Food Chem. 185 (2015) 430-436. [5] S. Kunath, M. Panagiotopoulou, J. Maximilien, N. Marchyk, J. Sänger, K. Haupt, Cell and tissue imaging with molecularly imprinted polymers as plastic antibody mimics, Adv Healthc Mater. 4 (2015) 1322-1326. [6] K. Rostamizadeh, M. Vahedpour, S. Bozorgi, Synthesis, characterization and evaluation of computationally designed nanoparticles of molecular imprinted polymers as drug delivery systems, Int J Pharmaceut. 424 (2012) 67-75. [7] D.R. Kryscio, N.A. Peppas, Critical review and perspective of macromolecularly imprinted polymers, Acta Biomater. 8 (2012) 461-473. [8] S. Li, S. Cao, M.J. Whitcombe, S.A. Piletsky, Size matters: Challenges in imprinting macromolecules, Prog Polym Sci. 39 (2014) 145-163. [9] D. Li, Y. Qin, H. Li, X. He, W. Li, Y. Zhang, A “turn-on” fluorescent receptor for detecting tyrosine phosphopeptide using the surface imprinting procedure and the epitope approach, Biosensors and Bioelectronics. 66 (2015) 224-230. [10] Y. Lv, T. Tan, F. Svec, Molecular imprinting of proteins in polymers attached to the surface of nanomaterials for selective recognition of biomacromolecules, Biotechnol Adv. 31 (2013) 1172-1186. [11] L. Qian, X. Hu, P. Guan, D. Wang, J. Li, C. Du, R. Song, C. Wang, W. Song, The effectively specific recognition of bovine serum albumin imprinted silica nanoparticles by utilizing a macromolecularly functional monomer to stabilize and imprint template, Anal Chim Acta. 884 (2015) 97-105. [12] X. Qu, F. Wang, Y. Sun, Y. Tian, R. Chen, X. Ma, C. Liu, Selective extraction of bioactive glycoprotein in neutral environment through Concanavalin a mediated template immobilization and dopamine surface imprinting, RSC Adv. 6 (2016) 86455-86463.
[13] S. Wang, Jin Ye, Z. Bie, Z. Liu, Affinity-tunable specific recognition of glycoproteins via boronate affinity-based controllable oriented surface imprinting, Chem Sci. 5 (2014) 1135-1140. [14] Y. Hao, R. Gao, D. Liu, B. Zhang, Y. Tang, Z. Guo, Preparation of biocompatible molecularly imprinted shell on superparamagnetic iron oxide nanoparticles for selective depletion of bovine hemoglobin in biological sample, J Colloid Interf Sci. 470 (2016) 100-107. [15] R. Gao, Y. Hao, L. Zhang, X. Cui, D. Liu, M. Zhang, Y. Tang, Y. Zheng, A facile method for protein imprinting on directly carboxyl-functionalized magnetic nanoparticles using non-covalent template immobilization strategy, Chem Eng J. 284 (2016) 139-148. [16] M. Sprynskyy, T. Kowalkowski, H. Tutu, E.M. Cukrowska, B. Buszewski, Ionic liquid modified diatomite as a new effective adsorbent for uranium ions removal from aqueous solution, Colloids and Surfaces A: Physicochemical and Engineering Aspects. 465 (2015) 159-167. [17] G. Shen, X. Zhang, Y. Shen, S. Zhang, L. Fang, One-step immobilization of antibodies for α-1-fetoprotein immunosensor based on dialdehyde cellulose/ionic liquid composite, Anal Biochem. 471 (2015) 38-43. [18] C.J. Carrasco, F. Montilla, L. Bobadilla, S. Ivanova, B.A.G. José Antonio Odriozola A, Oxodiperoxomolybdenum complex immobilized onto ionic liquid modified SBA-15 as an effective catalysis for sulfide oxidation to sulfoxides using hydrogen peroxide, Catal Today. 255 (2015) 102-108. [19] C. Du, X. Hu, P. Guan, Xumian Gao, R. Song, J. Li, L. Qian, N. Zhang, L. Guoa, Preparation of surface-imprinted microspheres effectively controlled by orientated template immobilization using highly cross-linked raspberry-like microspheres for the selective recognition of an immunostimulating peptide, J Mater Chem B. 4 (2016) 1510-1519. [20] S. Sasaki, T. Ooya, Y. Kitayama, T. Takeuchi, Molecularly imprinted protein recognition thin films constructed by controlled/living radical polymerization, J Biosci Bioeng. 119 (2015) 200-205. [21] S. Li, K. Yang, J. Liu, B. Jiang, L. Zhang, Y. Zhang, Surface-Imprinted nanoparticles prepared with a His-Tag-Anchored epitope as the template, Anal Chem. 87 (2015) 4617-4620. [22] X. Tang, W. Liu, J. Chen, J. Jia, Z. Ma, Q. Shi, Y. Gao, X. Wang, S. Xu, K. Wang, P. Guo, D. He, Preparation and evaluation of polydopamine imprinting layer coated multi-walled carbon nanotubes for the determination of testosterone in prostate cancer LNcap cells, Anal Methods-UK. 7 (2015) 8326-8334. [23] W. Wu, L. Yang, F. Zhao, B. Zeng, A vanillin electrochemical sensor based on molecularly imprinted poly(1-vinyl-3-octylimidazole hexafluoride phosphorus) − multi-walled carbon nanotubes@polydopamine – carboxyl single-walled carbon nanotubes composite, Sensors and Actuators B: Chemical. 239 (2017) 481-487. [24] G. Wu, J. Li, X. Qu, Y. Zhang, H. Hong, C. Liu, Template size matched film thickness for effectively in situ surface imprinting: A model study of glycoprotein imprints, RSC Advances. 5 (2015) 47010-47021. [25] M. Lin, H. Huang, Z. Liu, Y. Liu, J. Ge, Y. Fang, Growth–dissolution–regrowth transitions of Fe3O4 nanoparticles as building blocks for 3D magnetic nanoparticle clusters under hydrothermal conditions, Langmuir. 29 (2013) 15433-15441.
[26] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, Jr. J. A. Montgomery, J. E Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. R. Cossi, N. J. Millam, M. Knox Klene, J. B.Cross, V. Bakken, C. Adamo, J. Jaramillo, R. E. Stratmann Gomperts, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. Martin, R. L. Morokuma, V. G.Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, D. J. Fox, Gaussian. Inc.: Wallingford, CT, 2013. [27] G. Guan, S. Zhang, S. Liu, Y. Cai, M. Low, C. P. Teng, I. Y. Phang, Y. Cheng, K. L. Duei, B. M. Srinivasan, Y. Zheng, Y. Zhang, M. Han, Protein induces layer-by-layer exfoliation of transition metal dichalcogenides, Journal of the American Chemical Society. 137 (2015) 6152-6155. [28] W. Song, Y. Liu, L. Qian, L. Niu, L. Xiao, Y. Hou, Y. Wang, X. Fan, Hyperbranched polymeric ionic liquid with imidazolium backbones for highly efficient removal of anionic dyes, Chem Eng J. 287 (2016) 482-491. [29] Z. Zhang, F. Zhang, Q. Zhu, W. Zhao, B. Ma, Y. Ding, Magnetically separable polyoxometalate catalyst for the oxidation of dibenzothiophene with H2O2, J Colloid Interf Sci. 360 (2011) 189-194. [30] Y. Jiang, C. Guo, H. Xia, I. Mahmood, C. Liu, H. Liu, Magnetic nanoparticles supported ionic liquids for lipase immobilization: Enzyme activity in catalyzing esterification, Journal of Molecular Catalysis B: Enzymatic. 58 (2009) 103-109. [31] B. Gao, P. Guan, X. Hu, L. Qian, Q. Wang, L. Yang, D. Wang, The performance optimization and specific adsorption of L-phenylalanine imprinted polymers using 1-vinyl-3-carboxymethylimidazolium chloride as functional monomer, Designed Monomer and Polymers. 18 (2015) 185-198. [32] Y. Lin, F. Wang, Z. Zhang, J. Yang, Y. Wei, Polymer-supported ionic liquids: Synthesis, characterization and application in fuel desulfurization, Fuel. 116 (2014) 273-280. [33] S. Zhang, Y. Zhang, G. Bi, J. Liu, Z. Wang, Q. Xu, H. Xu, X. Li, Mussel-inspired polydopamine biopolymer decorated with magnetic nanoparticles for multiple pollutants removal, J Hazard Mater. 270 (2014) 27-34. [34] M. Müller, B. Torger, E. Bittrich, E. Kaul, L. Ionov, P. Uhlmann, M. Stamm, In-situ ATR-FTIR for characterization of thin biorelated polymer films, Thin Solid Films. 556 (2014) 1-8. [35] Y. Wang, S. Wang, H. Niu, Y. Ma, T. Zeng, Y. Cai, Z. Meng, Preparation of polydopamine coated Fe3O4 nanoparticles and their application for enrichment of polycyclic aromatic hydrocarbons from environmental water samples, J Chromatogr a. 1283 (2013) 20-26. [36] Y. Zhang, M. Zhang, J. Yang, L. Ding, J. Zheng, J. Xu, Facile synthesis of sea urchin-like magnetic copper silicate hollow spheres for efficient removal of hemoglobin in human blood, J Alloy Compd. 695 (2017) 3256-3266. [37] H. He, G. Fu, Y. Wang, Z. Chai, Y. Jiang, Z. Chen, Imprinting of protein over silica nanoparticles via surface graft
copolymerization using low monomer concentration, Biosensors and Bioelectronics. 26 (2010) 760-765. [38] A.K. Wright, M.R. Thompson, Hydrodynamic structure of bovine serum albumin determined by transient electric birefringence, Biophys J. 15(2 Pt 1) (1975) 137-141. [39] M. Zhang, Y. Wang, X. Jia, M. He, M. Xu, S. Yang, C. Zhang, The preparation of magnetic molecularly imprinted nanoparticles for the recognition of bovine hemoglobin, Talanta. 120 (2014) 376-385. [40] X. Li, B. Zhang, L. Tian, W. Li, H. Zhang, Q. Zhang, Improvement of recognition specificity of surface protein-imprinted
magnetic
microspheres
by
reducing
nonspecific
adsorption
of
competitors
using
2-methacryloyloxyethyl phosphorylcholine, Sensors and Actuators B: Chemical. 208 (2015) 559-568. [41] L. Qian, X. Hu, P. Guan, D. Wang, J. Li, C. Du, R. Song, C. Wang, W. Song, The effectively specific recognition of bovine serum albumin imprinted silica nanoparticles by utilizing a macromolecularly functional monomer to stabilize and imprint template, Anal Chim Acta. 884 (2015) 97-105. [42] Y. Liu, Z. Liu, J. Gao, J. Dai, J. Han, Y. Wang, J. Xie, Y. Yan, Selective adsorption behavior of Pb(II) by mesoporous silica SBA-15-supported Pb(II)-imprinted polymer based on surface molecularly imprinting technique, J Hazard Mater. 186 (2011) 197-205. [43] J. Fu, Z. Chen, M. Wang, S. Liu, J. Zhang, J. Zhang, R. Han, Q. Xu, Adsorption of methylene blue by a high-efficiency adsorbent (polydopamine microspheres): Kinetics, isotherm, thermodynamics and mechanism analysis, Chem Eng J. 259 (2015) 51-61. [44] C. Ho, S. Ding, The pH-controlled nanoparticles size of polydopamine for anti-cancer drug delivery, J Mater Sci: Mater Med. 24 (2013) 2381-2390. [45] Y. Yu, J.G. Shapter, R. Popelka-Filcoff, J.W. Bennett, A.V. Ellis, Copper removal using bio-inspired polydopamine coated natural zeolites, J Hazard Mater. 273 (2014) 174-182. [46] X. Li, B. Zhang, W. Li, X. Lei, X. Fan, L. Tian, H. Zhang, Q. Zhang, Preparation and characterization of bovine serum albumin surfaceimprinted thermosensitive magnetic polymer microsphere and its application for protein recognition, Biosensors and Bioelectronics. 51 (2014) 261-267. [47] D. G. Riveros, K. Cordova, C. Michiels, H. Verachtert, G. Derdelinckx, Polydopamine imprinted magnetic nanoparticles as a method to purify and detect class II hydrophobins from heterogeneous mixtures, Talanta. 160 (2016) 761-767.
Highlights Template immobilization was combined with surface imprinting technology. Ionic liquid functionalized Fe3O4 nanoparticles was utilized as substrate. Dopamine was chosen to prepare template-size tailored imprinted layers. The imprinted nanoparticle showed high adsorption capacity and excellent selectivity.
Graphical Abstract