Ni2+-BSA Directional Coordination-Assisted Magnetic Molecularly

Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Ni2+-BSA Directional Coordination-Assisted Magnetic Molecularly Imprinted Microspheres with Enhanced Specific Rebinding to Target Proteins Jingjing Zhou, Yufei Wang, Jun Bu, Baoliang Zhang,* and Qiuyu Zhang* MOE Key laboratory of Material Physics and Chemistry under Extraordinary Conditions, School of Science, Northwestern Polytechnical University, Xi’an 710072, P. R. China Downloaded via BUFFALO STATE on July 21, 2019 at 02:32:21 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

S Supporting Information *

ABSTRACT: Protein imprinting technology is of interest in drug delivery, biosensing, solid-phase extraction, and so forth. However, the efficient recognition and separation of proteins have remained challenging to date. Toward this, under the assistance of Ni2+-bovine serum albumin (BSA) directional coordination strategy, magnetic BSA-imprinted materials had been synthesized via dopamine self-polymerization on hollow Fe3O4@mSiO2 microspheres (mSiO2 referred as mesoporous silica). The well-defined imprinted microspheres possessed more satisfactory adsorption capacity (266.99 mg/g), enhanced imprinting factor (5.45), and fast adsorption saturation kinetics (40 min) for BSA, superior to many previous reports. Benefiting from the coordinate interaction between Ni2+ and BSA, these fabricated microspheres exhibited excellent specificity not only in individual and competitive protein rebinding samples but also in bovine serum. Combined with the directional coordination method, the magnetic-imprinted composite materials to selectively capture target proteins could provide promising potential in applications. KEYWORDS: molecularly imprinted polymers, directional coordination, dopamine, rebinding specificity, effective adsorption

1. INTRODUCTION

rebinding of proteins with corresponding feature fragment and surface imprinting to reduce mass transfer resistance. With multiepitopes as templates, Zhang et al.11 prepared the imprinted particles via poly(ether sulfone) self-assembly that could capture various target proteins at the same time with high selectivity, even in human plasma. Liu et al.12,13 developed the boronated affinity surface imprinting, allowing for the controllable-oriented preparation of MIPs to selective recognition for glycoproteins, glycans, and monosaccharides. Also, to gain more excellent protein adsorption abilities, porous materials provided with diverse pore structures were preferred to improve the efficient surface. Zhang et al.14 utilized fibrous SiO2 microspheres as matrix to prepare glutathione (GSH)imprinted thermosensitive particles, which exhibited good adsorption and selectivity for GSH. Shi et al.15 chose large-

Aroused from the biosystem of antigen−antibody, molecularly imprinted polymers (MIPs), the next-generation artificial polymer receptors, have been developed through polymerization of proper functional monomers and cross-linking agents in the presence of templates.1,2 After the elution treatment, imprinting cavities possessing functionally and sterically complementary affinity toward templates are left behind.3,4 To date, MIPs have been applied in wide areas, for instance, chemical sensors,5,6 solid-phase extractions,7 immunoassays,8 and drug delivery.9 Nonetheless, the boom of relevant studies on MIPs is associated with small molecules. Macromolecules, especially proteins, face many challenges in the imprinting technique.10 Despite great efforts, the adsorption and recognition performances of MIPs still lag behind due to the bulky structures, flexible molecular conformation, and complex surface of proteins. Many imprinting strategies were proposed to surmount the obstacles, for example, epitope technology to realize the © XXXX American Chemical Society

Received: April 14, 2019 Accepted: June 27, 2019 Published: June 27, 2019 A

DOI: 10.1021/acsami.9b06507 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

mechanical stirring. After maintaining the stirring state for 0.5 h at 25 °C, all of these reactants were heated to 70 °C. When the reaction was kept for 16 h, the resultant sample was then refluxed at 60 °C overnight in alcoholic solution of ammonium nitrate to extract CTAB. After the wash treatment with water and lyophilization, the hollow Fe3O4@mSiO2 (denoted HMMS) microspheres were prepared at last. 2.2.2. Synthesis of HMMS@Ni2+-BSA-Imprinted Polydopamine (PDA) Microspheres. In this work, BSA was chosen as the template protein for imprinting. Directional coordination: Before BSA imprinting, HMMS microspheres were modified with amino groups (−NH2) and subsequently complexed with Ni2+. Typically, 50 mg of HMMS was dispersed into the solution mixed with 40 mL of ethanol, 40 mL of water, 0.5 mL of ammonia aqueous solution (25 wt %), and 1.0 mL of APTES. After stirring overnight at 70 °C, the microspheres modified with −NH2 (defined HMMS−NH2) were rinsed with ethanol and water respectively for several times. Subsequently, all of these microspheres were added into 50 mL of water containing 1.0 g of nickel nitrate hexahydrate, refluxed for 10 h at 80 °C, and collected with a magnet at last. Imprinting polymerization: The above-obtained microspheres were dispersed into Tris−HCl buffer (pH = 8.0, 50 mM/mL, 20 mL) containing 20 mg of BSA. After stirring for 0.5 h, 5.0 mL of Tris−HCl buffer containing 70 mg of dopamine was injected into the above solution at 30 °C.23,24 After maintaining the polymerization over 24 h, the prepared Ni2+-BSA-imprinted microspheres were collected with a magnet and washed with water to get rid of the remaining oligomers. Afterward, all of the samples were eluted with acetic acid solution (6.0 vt %) for the removal of the template protein. These resultant HMMS@Ni2+-BSA-imprinted polydopamine (PDA) microspheres (denoted HMMS@Ni2+-MIPs) were lyophilized for the subsequent investigations. Correspondingly, the nonimprinted ones (denoted HMMS@Ni2+-nonimprinted polymers (NIPs)) were prepared with the same procedures only in the absence of the template protein BSA. 2.3. Adsorption Experiments. Target proteins were incubated at 30 °C for 2 h with 5.0 mg of HMMS@Ni2+-MIPs or HMMS@Ni2+NIPs microspheres dispersed in 6.0 mL of aqueous solution. With the aid of a UV-2550 spectrophotometer, the protein content was determined at 596 nm according to the Bradford assay25 (see the Supporting Information) after separating the microspheres with a magnet.

pore silica particles as matrixes to accomplish BSA imprinting, and the high binding capacities and fast adsorption kinetics were received for the well-designed porous structures. Moreover, researchers also dedicated themselves to explore more appropriate strategies to improve the rebinding specificity of MIPs. Hu et al.16 designed a promising kind of cyclodextrin-based ionic liquid as the functional monomer to strengthen the affinity between substrates and template molecules. The prepared imprinting particles thus exhibited good recognition ability to cytochrome C (Cyt C). Li et al.17,18 grafted poly(2-methacryloyloxyethyl phosphorylcholine) chains on the surface of the MIP shell by activators generated by electron transfer−atom transfer radical polymerization and acquired satisfactory anti-nonspecific adsorption performances. Although the above-mentioned technologies provided a potential prospect targeting the imprinting performances, more efforts should be committed for increasing the quality of protein imprinting in terms of adsorption efficiency and rebinding specificity simultaneously. To our knowledge, the coordination between metal ions and proteins, one of the noncovalent effects, was rather stable, which made protein imprinting technology more viable and effective.19−21 Inspired by this, we put forward a more feasible method, Ni2+-BSA directional coordination, into the magnetic imprinting system for the efficient recognition of BSA. First, hollow Fe3O4 microspheres with prominent monodispersibility were selected to serve as substrates for accelerating the separation of imprinted microspheres under an external magnetic field. Moreover, aiming to complex more Ni2+, a layer of mesoporous SiO2 modified with amino groups was coated on their surface. Afterward, the template protein BSA could be trapped sufficiently by Ni2+ via coordinate interaction and immobilized in imprinting cavities through dopamine selfpolymerization. Based on this approach, the performances were enhanced in terms of both the adsorption capacity and rebinding specificity.

Q = (C0 − C)V /m

2. EXPERIMENTAL SECTION

(1)

Here, formula 1 was defined to calculate the adsorption capacities (Q, mg/g) of HMMS@Ni2+-MIPs or HMMS@Ni2+-NIPs to target proteins. C0 is the initial concentration of protein solution (mg/ mL), C is the protein concentration in supernatants after adsorption (mg/mL), V is the volume of protein solution (mL), and m is the mass of HMMS@Ni2+-MIPs or HMMS@Ni2+-NIPs (g). IF and selectivity factor (β) were used to evaluate the rebinding specificity of HMMS@Ni2+-MIPs or HMMS@Ni2+-NIPs to target proteins and derived as follows

2.1. Materials. Sodium acetate (anhydrous), ferric chloride hexahydrate (FeCl3·6H2O), sodium polyacrylate, sodium hydroxide (NaOH), trisodium citrate, and nickel nitrate hexahydrate were obtained from Shanghai Chemical Reagents Company (China). Cyclohexane, 3-aminopropyltriethoxysilane (APTES), isopropanol (IPA), and tetraethyl orthosilicate (TEOS) were supplied by Guangdong Chemical Reagents Engineering-Technological Research and Development Center (China). Cetyltrimethylammonium bromide (CTAB) and urea were obtained from Sinopharm Chemical Reagent Co., Ltd. (China). Dopamine hydrochloride was purchased from Sigma-Aldrich (Tokyo, Japan). Ammonium persulfate, sodium tartrate dibasic dihydrate, and dimethylglyoxime were obtained from Shanghai Macklin Biochemical Co. Ltd. (China). Hydrochloric acid (HCl), Coomassie brilliant blue G-250, and tris(hydroxymethyl) aminomethane (Tris) were bought from Shanghai Shan Pu Chemical Co. Ltd. (China). BSA, bovine hemoglobin (BHb), Cyt C, ovalbumin (OVA), ribonuclease A (RNase A), and lysozyme (Lyz) were purchased from Amresco (Solon, OH). 2.2. Preparation of Hollow Fe3O4@mSiO2@Ni2+-BSA-Imprinted Polydopamine Microspheres. 2.2.1. Synthesis of Hollow Fe3O4@mSiO2 Microspheres. First, the hollow Fe3O4 microspheres were fabricated with the hydrothermal method.22 Subsequently, the mesoporous SiO2 layer was coated around hollow Fe3O4 microspheres in a typical biliquid phase synthesis: 0.50 g of hollow Fe3O4 microspheres was dispersed in the water (30 mL) containing 0.6 g of urea and 1.0 g of CTAB to form a suspension liquid. Afterward, 1.0 mL of IPA and 30 mL of cyclohexane were injected into the mixture. Then, 1.2 mL of TEOS was added dropwise under vigorous

IF = Q MIP/Q NIP

(2)

β = IFBSA /IFnon ‐ BSA

(3)

In formula 2, QMIP and QNIP represent the adsorption capacities of target proteins on HMMS@Ni2+-MIPs and HMMS@Ni2+-NIPs, respectively. In formula 3, IFBSA and IFnon‑BSA are applied to represent the imprinting factors (IFs) to the template protein BSA and nonBSA protein, respectively.26 2.4. Characterization. A transmission electron microscope (TEM, JEOL JEM-3010) was used to investigate the morphology of all of these prepared microspheres. Fourier transform infrared (FTIR) spectra were received by testing the sample compact mixed with KBr in a TENSOR27 FTIR spectrometer (Bruker). Furthermore, the polymer content of HMMS@Ni2+-MIPs and HMMS@ Ni2+-NIPs was examined with the aid of thermogravimetric analysis (TGA, Q50, TA Instruments) through heating from 35 to 900 °C in an oxygen atmosphere. A vibrating sample magnetometer (VSM, LakeShore 7307) was chosen to assess the magnetic properties of all B


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Figure 1. Scheme of synthesis of HMMS@Ni2+-MIPs. of the products. In addition, a UV-2550 (Shimadzu) spectrophotometer was applied to measure the protein content of the solution at 596 nm. Moreover, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used for the qualitative tests of protein adsorption capacity for imprinted microspheres after staining the protein fragments with the agent Coomassie Brilliant Blue G-250.

3. RESULTS AND DISCUSSION 3.1. Synthesis. Regarding specific rebinding, its insufficiency inhibited the practical applications of protein imprinting getting booming.2,27 Therefore, a novel method, “directional coordination”, which complexed Ni2+ with BSA and further accomplished imprinting, was proposed to ameliorate the issue. As depicted in Figure 1, hollow Fe3O4 microspheres, possessing excellent water monodispersity and uniform particle size compared to those of solid ones, were selected as substrates. By the surfactant-templated sol−gel approach, a layer of the CTAB/SiO2 composite with a mesostructure was coated on the hollow Fe3O4 microspheres, utilizing TEOS and CTAB as the silica precursor and structure-directing agent, respectively.28 After removing CTAB template molecules, the HMMS microspheres were thus fabricated. Then, the amino groups were modified onto the surface of HMMS microspheres with APTES hydrolysis, for the following complexation of Ni2+. Prior to imprinting, first, the template protein BSA was chelated with Ni2+. Subsequently, the imprinting polymerization was achieved in the presence of dopamine. The resultant HMMS@Ni2+-MIPs were finally received by elution of template proteins. Also, according to above procedures, the comparative microspheres HMMS@Ni2+-NIPs were fabricated only in the absence of the template protein BSA.26 3.2. Characterization. The typical morphologies of hollow Fe3O4, HMMS, HMMS@Ni2+-MIPs, and HMMS@Ni2+-NIPs were exemplified in the TEM images of Figure 2. From Figure 2a, the hollow Fe3O4 microspheres possessed an average diameter of ∼200 nm. As a result of the chelating effect from citrate groups, the obtained hollow Fe3O4 microspheres would be stabilized and own good water monodispersibility,29 which was beneficial to the surface modification by solution-phase synthesis. Via the surfactant-templated sol−gel approach, TEOS would be hydrolyzed into silicate oligomers and further coassembled with CTAB in the presence of hollow Fe3O4 as seeds to generate HMMS microspheres after the extraction of surfactants. As shown in Figure 2b, HMMS microspheres exhibited an obvious core−shell structure and the mesoporous

Figure 2. TEM images of hollow Fe3O4 (a), HMMS microspheres (b), HMMS@Ni2+-MIPs (c), and HMMS@Ni2+-NIPs (d).

silica shell had a mean thickness of ∼45 nm. In addition, it is worth noting that the good water monodispersibility of HMMS microspheres maintained, favorable for the succeeding BSA imprinting. Dopamine could be oxidized by the oxygen in weak alkaline aqueous solution, which in turn initiated selfpolymerization.30 From Figure 2c, HMMS@Ni2+-MIPs microspheres were successfully prepared with dopamine and BSA as the monomer and template protein, respectively. It was simple to distinguish HMMS@Ni2+-MIPs from HMMS due to the thicker composite shell (∼60 nm) of mesoporous silica and polymer. Especially, the outer layer of HMMS@Ni2+-NIPs (Figure 2d) was similar to that of HMMS@Ni2+-MIPs in thickness, but the morphology was slightly different. We thought the involvement of BSA would affect the polymerization process, further leading to the structural dissimilarity. The characteristic peaks of Fe3O4, SiO2, and PDA, such as Fe−O, Si−O−Si, C6H6, N−H, and O−H, were identified in Figure S1a (see the Supporting Information). From Figure 3a, the representative type IV curve was exhibited in the N2 C


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Figure 3. N2 adsorption−desorption isotherms (a) and pore size distribution profile (b) of HMMS.

Figure 4. Isothermal adsorption equilibrium tests (a) and adsorption kinetic curves (b) of BSA on HMMS@Ni2+-MIPs and HMMS@Ni2+-NIPs.

3.3. Adsorption Properties. With respect to the saturated adsorption performances, an extensive investigation was carried out for a wide concentration range (0.0−1.4 mg/mL) and in response to the equilibration time of 2 h in our previous reports.2,18 From Figure 4, the adsorption capacity of HMMS@Ni2+-MIPs to BSA increased rapidly until the concentration come up to 0.8 mg/mL, since quantities of imprinting cavities being vacant for BSA adsorption; and the BSA binding assay remained saturated from 0.8 to 1.4 mg/mL, the same tendency as that for HMMS@Ni2+-NIPs. Moreover, it was evident that the Langmuir isotherm model fitted better than the Freundlich model in the range of 0.0−1.4 mg/mL, in the context of its correlation coefficient, R2, more proximate to 1.00 (Tables S1 and S2). It suggested that the binding sites on HMMS@Ni2+-MIPs were monolayers and homogeneous. On the basis of above results and comparison, the optimum initial BSA concentration in the following experiments was determined as 0.8 mg/mL. Due to the presence of the magnetic hollow structure and silica mesopores, small molecules like H2O would penetrate into the internal cavity through MIPs. Hence, a diffusion force was generated from the protein concentration difference between the outer and inner solutions of the microspheres, which further realized the rapid adsorption and recognition of target proteins. Hence, we turned to the kinetics tests to verify the above assumption. The faster adsorption equilibration (40 min) was observed (Figure 4b) in comparison with our

adsorption−desorption isotherm of HMMS microspheres according to the International Union of Pure and Applied Chemistry nomenclature. The apparent hysteresis loop in the p/p0 range of 0.5−0.8 and a sharp capillary condensation step indicated the characteristics of HMMS microspheres with narrow pore size distribution.31 Also, the mesopore size of HMMS was ∼6.75 nm from the curve of pore size distribution (Figure 3b), which descended from the desorption branch. Furthermore, the total pore volume and the Brunauer− Emmett−Teller surface area of HMMS were measured as 0.52 cm3/g and ∼303 m2/g, respectively. The excellent pore structure and high specific surface area would be favorable for the complexation of Ni2+. Combined with thermogravimetric analysis (Figure S1b), the weight losses of 56.23 and 46.89 wt % were assigned to the polymer degradation in MIPs and NIPs. Mesoporous silica and imprinted polymers were believed to be successfully coated on hollow Fe3O4 microspheres. In addition, magnetic properties (Figure S1c) of these four kinds of microspheres were characterized at room temperature and their saturation magnetization values were examined as 93.0, 80.4, 41.9, and 54.6 emu/g, respectively, which enabled these microspheres to be separated rapidly (Figure S1d). On the base of above results, hollow Fe3O4, HMMS, HMMS@Ni2+-MIPs and HMMS@Ni2+-NIPs microspheres were verified to be prepared successfully. D


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Figure 5. Adsorption abilities of different microspheres to BSA (a) and effect of Ni2+ quantitative coordination on binding performances (b).

previous works.18,32 The diffusion force made BSA more accessible to imprinting sites, which enhanced the protein binding efficiency. Fan et al.33 constructed a novel method of rattle-type imprinted nanospheres with a magnetic hollow structure to improve the binding performances. The similar concept and rule were consistent with this work. Based on the above analysis, the initial concentration of BSA solution and the equilibrated adsorption time were determined as 0.8 mg/mL and 40 min, respectively, to carry out the following experiments. 3.4. Effect of Directional Coordination. It was of interest to put forward the approach directional coordination into the imprinting system based on the preliminary results. As we know, the protein, formed by dehydration condensation of amino acids, carried a plenty of −NH2 and −OH groups, whose N and O atoms possessed lone-pair electrons. The template proteins possessing these lone-pair electrons could be trapped by Ni2+ due to the strong interaction with d orbitals of ions. This would ensure the template proteins to be confined in imprinting cavities via polymerization.34 In this regard, a sequence of control assay and ancillary investigation were conducted as follows. Firstly, the interaction between Ni2+ with −NH2 groups on HMMS−NH 2 and HMMS@Ni 2+ -MIPs was confirmed through FTIR spectroscopy (Figure S2). To our knowledge, the N−H scissoring vibration was assigned to the peaks at ∼1620 cm-1. Both of the position shifts and intensity changes of the peaks were sufficient to prove the interaction of Ni2+ with −NH2 groups.34 According to the previous report, numerous −OH groups generated on PDA after the noncovalent self-assembly and covalent polymerization of dopamine.30 On the one hand, BSA was immobilized with PDA directly on HMMS microspheres (defined as HMMS@MIPs) to get rid of the interaction between −OH (on PDA) and −NH 2 (from APTES hydrolysis). The morphology and rebinding performances were separately given in Figures S3 and5a. Without complexing with Ni2+, the specific recognition ability of HMMS@MIPs to BSA (IF = 1.58) declined significantly compared to that of HMMS@Ni2+-MIPs (IF = 5.45), indicating the dominant role of Ni2+-BSA coordinate interaction preliminarily. Interestingly, when mentioned the adsorption ability to BSA, HMMS@Ni2+NIPs (Q = 48.96 mg/g) were weaker than HMMS@NIPs (Q = 87.98 mg/g). Since some −OH groups on PDA would complex with Ni2+ during the non-BSA polymerization

process, it was considered that there were a less number of −OH groups on the surface of HMMS@Ni2+-NIPs for capturing BSA via hydrogen bonding and electrostatic interaction, leading to the weaker nonspecific adsorption. On the other hand, when taking into account the hydrogen bonding between −OH (on PDA) and −NH2 (from APTES hydrolysis), another control-imprinted microspheres based on HMMS−NH2 were prepared and defined as HMMS@Ni2+MIPs-0.00 (Figure 5b). Nevertheless, their adsorption and rebinding performances (Q = 85.26 mg/g, IF = 1.38) were not as good as those of HMMS@MIPs. The reason might be that the excess APTES was added when modifying amine groups onto HMMS in synthesis procedures, and plentiful −NH2 groups would form hydrogen bonds with −OH on PDA in succeeding imprinting polymerization. Thus, the amount of effective functional groups distributed in BSA imprinting sites reduced, causing the decrease in binding capacities. Besides, this control analysis also demonstrated that the satisfactory rebinding properties were attributed to Ni2+-BSA directional coordination rather than the electrostatic interaction of −NH2 (on HMMS−NH2) with BSA. Apart from the above analysis, the microspheres of HMMS− NH2 complexed with Ni2+ (denoted HMMS−NH2/Ni2+) were fortuitously discovered to possess even better adsorption ability to BSA (165.85 mg/g, Figure 5a, blue bar) than HMMS@MIPs. This would benefit from the coordination between Ni2+ with BSA, indicating the vital role of Ni2+ in this system as well. To further determine the effect of Ni2+-BSA directional coordination, a Ni2+ quantitative complexed analysis had been examined, as illustrated in Figure 5b. Herein, a certain amount of Ni2+ was complexed by HMMS−NH2; afterward, BSA was coordinated with Ni2+ and then frozen by dopamine polymerization on the substrates. These microspheres were defined as HMMS@Ni2+-MIPs-x (x, complexation amount of Ni2+, mmol Ni2+/g HMMS, see the Supporting Information). Among them, HMMS@Ni 2+ -MIPs-2.11 was equal to HMMS@Ni2+-MIPs in the above description. From Figure 5b, the complexation amount of Ni2+ was positively correlated not only with the adsorption capacity of MIPs but also with IF. The phenomenon was very interesting, and we gave the corresponding explanation as follows. With the increase of complexed Ni2+, more template proteins would bind onto the microspheres to produce more efficient imprinting sites on HMMS@Ni2+-MIPs-x, and in turn to rebind more BSA E


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Table 1. Quantitative Comparison of QMIP, IF, and Adsorption Equilibrium Time for Protein-Imprinted Particlesa protein-imprinted particles

QMIP (mg/g)

IF

equilibrium time (h)

ref

large-pore SiO2@BSA-p(APTES-co-OTMS) Fe3O4@SiO2@BSA-PDA@MPC γ-Fe2O3@BSA-pMBA PGMA/PS@BSA-PDA Fe3O4@void@BSA-pIL SiO2@Cu2+@PSA-p(MAA-co-HEA) HMMS@Ni2+-MIPs

162.82 8.26 300.00 72.7 130.19 7.7 266.99

3.03 5.74

1 2 24 2 1 0.33 0.67

201715 201718 201735 201824 201833 201536 This work

4.60 2.79 2.60 5.45

a

OTMS, octyltrimethoxysilane; MPC, 2-methacryloyloxyethyl phosphorylcholine; MBA, N,N-methylenebisacrylamide; IL, ionic liquid; MAA, methacrylic acid; and HEA, 2-hydroxyethyl methacrylate.

Figure 6. Individual rebinding specificity (a) and competitive adsorption performances (b−d) of HMMS@Ni2+-MIPs. Quantitative rebinding selective performance in the protein mixture (CBSA = CBHb = 0.5 mg/mL, Tris−HCl buffer, pH = 8.0) (b). Qualitative competitive adsorption analysis for protein mixed solution (CBSA = CLyz = 0.5 mg/mL, Tris−HCl buffer, pH = 8.0) with SDS-PAGE (c): lane 1, markers; lane 2, protein mixed solution; lane 3, protein mixed solution after adsorption by HMMS@Ni2+-MIPs; and lane 4, protein eluate from HMMS@Ni2+-MIPs. Adsorption for fetal bovine serum (FBS) (d): lane 1, markers; lane 2, 10-fold diluted FBS; lane 3, 10-fold diluted FBS after adsorption by HMMS@ Ni2+-MIPs; and lane 4, protein eluate from HMMS@Ni2+-MIPs. As for SDS-PAGE, 6.0 mg of HMMS@Ni2+-MIPs was added for adsorption; the injected amount of protein solution was 10 μL.

impressive. Hereby, we made a quantitative comparison of QMIP, IF, and adsorption equilibrium time on proteinimprinted particles (Table 1). Hollow Fe3O4 microspheres, the substrates, provided with outstanding properties, such as good water monodispersity, hollow structure, and light weight, result in the faster adsorption kinetics and lower mass transfer resistance to BSA compared to those for the solid ones.18,33,35 Additionally, more accessibility of target proteins to the efficient imprinting sites was obtained for the well-designed pore structure and high specific surface area.15,24 Also, as given in Table 1, BSA-PDA-imprinted materials exhibited satisfactory rebinding specificity18,24 in the imprinting systems, as well as in this work. When taking into account the metal-ion

molecules in the protein adsorption process. Meanwhile, when the Ni2+ complexation amount was increased, more −OH groups on PDA of HMMS@Ni2+-NIPs-x would be involved to form coordinate bonds with Ni2+; in other words, fewer −OH groups remained to accomplish nonspecific adsorption, making the rebinding ability of HMMS@Ni2+-NIPs-x to decrease slightly. On the basis of these analyses, it was believed that the novel strategy of Ni2+-BSA directional coordination held great importance for the specific rebinding property of HMMS@ Ni2+-MIPs. 3.5. Comparisons. Once combining Ni2+-BSA directional coordination with hollow magnetic microspheres, the effect on the imprinting performances of HMMS@Ni2+-MIPs was F


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ACS Applied Materials & Interfaces coordination, Zhang et al.36 synthesized the porcine serum albumin (PSA)-imprinted polymers on silica beads with the Cu2+ chelating method. Although the adsorption equilibrium was faster (20 min), both of IF (2.60) and rebinding capacity (7.7 mg/g) were still lagging behind compared to those of HMMS@Ni2+-MIPs. Upon a comprehensive evaluation of QMIP, IF, and adsorption equilibrium time, it was evident that HMMS@Ni2+-MIPs acquired the best performance in these protein-imprinted particles. The elegant imprinting strategy assisted with Ni2+-BSA directional coordination was further verified to be advantageous. 3.6. Rebinding Specificity. Rebinding specificity is of importance to evaluate the worth of imprinted materials.37,38 In accordance with the molecule weight (MW) and isoelectric point (pI) of BSA (MW 68 kDa, pI 4.9), a range of proteins were selected as analogues in this work and the specific rebinding properties had been examined (Figure 6a). In these analogues, OVA (MW 43 kDa, pI 4.7) and BHb (MW 67 kDa, pI 6.8) were negative in Tris−HCl buffer (pH = 8.0, 50 mM/ mL), as the same electricality as that of BSA, which led to that their adsorption capacities were correspondingly higher than that of other analogues. Because of the lower MW and an approximate pI to that of BSA, OVA obtained IFOVA of 1.16 and β of 4.70, suggesting the poor affinity to BSA-imprinted cavities in the spatial structure. Moreover, compared to those of the template protein BSA, BHb possessed a similar molecular scale and a higher pI, but it displayed IFBHb of 1.09 and β of 5.00. This means that BHb could not be recognized properly. As depicted in Figure 6a, RNase A (MW 12.6 kDa, pI 7.8) acquired the lowest adsorption performance (IFRNase A = 1.23, β = 4.43). Although RNase A was small enough for mass transfer, the pH of Tris−HCl buffer was approximate to its pI, making the protein solubility not optimal. Otherwise, Lyz (MW 14.4 kDa, pI 10.8) and Cyt C (MW 10.5 kDa, pI 12.5) provided higher pIs than that of BSA. Clearly, both of them were positive in Tris−HCl buffer. BSAimprinted cavities could not retain these analogues efficiently due to the discrepant electrical behaviors in aqueous solution. Briefly, according to the above analysis, BSA-imprinted cavities on HMMS@Ni2+-MIPs exhibited satisfactory affinity, and their excellent individual rebinding specificity to the template molecule BSA was also verified. Furthermore, the protein competitive adsorption performances of HMMS@Ni2+-MIPs were investigated via quantitative and qualitative tests. Here, the quantitative analysis was shown in Figure 6b. Despite the decrease of the rebinding capacity, HMMS@Ni2+-MIPs exhibited favorable rebinding selectivity (β = 4.14) in the protein mixture of BSA and BHb (CBSA = CBHb = 0.5 mg/mL, Tris−HCl buffer, pH = 8.0). With regard to the qualitative trial, SDS-PAGE was aided to demonstrate the selective properties of HMMS@Ni2+-MIPs. As illustrate in Figure 6c, lane 2 revealed the protein mixed solution containing BSA and Lyz (CBSA = CLyz = 0.5 mg/mL, Tris− HCl buffer, pH = 8.0). When adsorbed with 6.0 mg of HMMS@Ni2+-MIPs, the protein eluate was obtained after treating these microspheres with acetic acid solution (6.0 vt %) just once. BSA reappeared in the protein eluate lane (lane 4), but the staining of Lyz was much weaker, suggesting the higher content of BSA. Besides, the adsorption property to fetal bovine serum (FBS) was presented in Figure 6d. There were few changes between lane 2 and lane 3 as the major protein was BSA in FBS. Afterward, BSA was thought to reappear in the eluate (Lane 4). According to the SDS-PAGE tests, the

rebinding specificity of HMMS@Ni2+-MIPs to BSA was further confirmed. 3.7. Reusability. It was vital to investigate the reusability of imprinted materials for the practical application.5,21,39 Hereby, the reusability of HMMS@Ni2+-MIPs for five adsorption− desorption cycles was examined, as given in Figure 7.

Figure 7. Reusability of HMMS@Ni2+-MIPs for BSA adsorption.

Obviously, the adsorption ability of HMMS@Ni2+-MIPs to BSA decreased gradually and ∼26.87% of adsorption capacity was totally lost after 5 cycles, which might be caused by the damage of imprinting cavities during the eluting procedures.26,40 Furthermore, the involvement of Ni2+-BSA directional coordination enhanced the imprinting performances as mentioned previously. Noncovalent interaction between Ni2+ and −NH2 or BSA was much weaker than the covalent one, and repeated washing operation would elute some Ni2+ away, in turn leading to the loss of adsorption to BSA. Therefore, HMMS@Ni2+-MIPs should be ameliorated for better reusability if applied in practical ways.

4. CONCLUSIONS In summary, a novel kind of Ni2+-BSA directional coordination-assisted magnetic-imprinted microsphere was successfully fabricated to improve both the effective adsorption and specific rebinding to BSA simultaneously. Ni2+ was proved to be vital for the imprinting performances, in which the complexation amount of Ni2+ was positively correlated not only with the adsorption capacity of MIPs but also with IF in the range of examination. Also, the optimal BSA adsorption ability (Q) and selective rebinding capability (IF) of HMMS@Ni2+-MIPs were determined as 266.99 mg/g and 5.45, respectively. As a result of the presence of a magnetic hollow structure and silica mesopores, the adsorption to BSA would come up to equilibrium efficiently (40 min), driven by the diffusion force generated from the protein concentration difference between the outer and inner solutions of the microspheres. Furthermore, HMMS@Ni2+-MIPs provided with excellent rebinding specificity to BSA according to the protein individual and competitive rebinding analyses, and the separation of BSA in bovine serum was also acceptable with the aid of the SDSPAGE test. As for reusability, HMMS@Ni2+-MIPs needed to be optimized for more stability since ∼26.87% of adsorption capacity was totally lost after 5 adsorption−elution cycles. Based on all of these results, this proposed Ni2+-BSA directional coordination-assisted strategy supplied a facile G


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approach for efficient adsorption and specific rebinding for proteins, holding great values for applications.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b06507. Experimental procedure of the Bradford assay; determination method of Ni2+ complexation amount; determination of protein quantitative competitive rebinding capacities; FTIR spectra, TGA and VSM analyses of hollow Fe 3 O 4 , HMMS, HMMS@Ni 2+ -MIPs, and HMMS@Ni2+-NIPs; liner fitting of Langmuir and Freundlich isotherm models; FTIR and partial enlargement of ∼1620 c/m spectra of HMMS−NH2, HMMS− NH2/Ni2+, and HMMS@Ni2+-MIPs; and TEM images of HMMS@MIPs and HMMS@NIPs (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (B.Z.). *E-mail: [email protected] (Q.Z.). ORCID

Baoliang Zhang: 0000-0002-0290-4949 Qiuyu Zhang: 0000-0002-4823-5031 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for the financial support provided by the State Key Program of National Natural Science Foundation of China (Grant no. 51433008), the National Natural Science Foundation of China (Grant no. 21704084), the International Cooperation and Exchanges NSFC (Grant no. 51711530233), and the Fundamental Research Funds for the Central Universities (3102017jc01001).

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Ni2+-BSA Directional Coordination-Assisted Magnetic Molecularly

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