Polyoxometalate-Coated Magnetic Nanospheres for Highly Selective

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Biological and Medical Applications of Materials and Interfaces

Polyoxometalate Coated Magnetic Nanospheres for Highly Selective Isolation of Immunoglobulin G Dandan Zhang, Zhiyong Guo, Pengfei Guo, Xue Hu, Xuwei Chen, and Jian-Hua Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b05334 • Publication Date (Web): 08 Jun 2018 Downloaded from http://pubs.acs.org on June 8, 2018

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ACS Applied Materials & Interfaces

Polyoxometalate Coated Magnetic Nanospheres for Highly Selective Isolation of Immunoglobulin G Dan-Dan Zhang, Zhi-Yong Guo, Peng-Fei Guo, Xue Hu, Xu-Wei Chen*, Jian-Hua Wang* Department of Chemistry, College of Science, Northeastern University, Shenyang 110819, China

Keywords: polyoxometalate; magnetic nanospheres; hydrogen-bonding interaction; electrostatic interaction; immunoglobulin G; isolation; human serum.

Abstract: Polyoxometalate [{a-PW11O39Zr(µ-OH)(H2O)}2]8- (POM1) is firstly prepared by sandwiching ZrIV among 2 mono-lacunary α-Keggin polyoxometalates, then novel magnetic nanoparticles (NPs) Fe3O4@PEI@POM1 are fabricated by coating POM1 onto the surface of magnetic Fe3O4@PEI NPs under electrostatic interaction. The obtained Fe3O4@PEI@POM1 NPs are characterized by FT-IR, zeta potential, VSM, TEM, EDXS and XRD. Ascribed to the hydrogen-bonding and electrostatic interactions, the NPs exhibit high adsorption selectivity towards IgG, and the adsorption capacity is high up to 304 mg g-1 under optimal adsorption conditions. By using 0.01% CTAB to strip the adsorbed protein specie, an elution efficiency of 95% is achieved. The feasibility of Fe3O4@PEI@POM1 NPs in real-world sample assay has been demonstrated by the selectively isolation of IgG heavy chain and light chain from human serum, as confirmed by SDS-PAGE assay.

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INTRODUCTION Immunoglobulins are attributed to the high abundant protein species in human or animal serum.1 As a critical section of the immune system binding humoral and cellular immunity, this large number of proteins is an enormous benefit for living body as their ability to kill the infected cells or tumor cells, and their important role in non-inflammatory opsonization and neutralization, and anti-inflammatory functions as well. There are five isotypes for immunoglobulin, i.e., IgG, IgA, IgM, IgD and IgE. IgG is the main immunoglobulin isotype in animal serum as well as in human serum, whose content is up to 75 % of the immunoglobulins. IgG could trigger immune signal by connecting with cellular receptors specific for the antibodies’ Fc section, enhance the visibility of the pathogen through opsonization and activate the complement system, therefore, IgG play an important role on the protecting the living body against the infiltrating of epithelial and mucosal barriers by bacteria and viruses.2,3 The extracted IgG from serum sample has been gaining extensive applications in medical and biological research,4 clinical diagnosis5 and industries.6 Generally, the extraction of IgG from serum are usually achieved by caprylic acid/saturated sulfate precipitation, followed with ensuing chromatography separation such as affinity chromatography7 or ion-exchange chromatography.8,9 Recently, solid phase extraction exploiting functional materials as the sorbents have been gaining increasing attention in IgG isolation due to its favorite efficiency and facile manipulations.10 Polyoxometalates (POMs) is a great family of metal-oxygen anionic clusters. The merits of reversible redox activity, large sizes, high negative charges and cage


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structure gain POMs wide applications in various fields, especially in the field of catalysis. Metal-substituted POMs prepared via the integration of external metal ions into the metal-oxygen frameworks of POMs not only provide efficient homogeneous catalysts for many reactions including H2O2-based epoxidation of olefins,11 oxygenation reactions of thioethers,12 Mukaiyama aldol and Mannich-type addition reactions13 and the cyclization of citronellal,14 but also could be used as a favorable substitute in protein hydrolysis. For instance, highly Lewis acidic metal ions CeIV or ZrIV substituted POMs have been proved to be excellent catalysts for peptide-bond or phosphoester-bond hydrolysis in oligopeptides,15 proteins16-19 and DNA.20 Moreover, ZrIV-substituted Keggin-type, Lindqvist-type and Wells-Dawson-type POMs exhibit hydrolysis specificity towards myoglobin at Asp-X peptide bonds, due to the electrostatic interactions between POMs and specific protein structural domain.21 Parac-Vogt et al demonstrate that POMs clusters are not only potential as artificial proteases for proteins hydrolysis, but also could be assembled into peptide chains as inorganic amino acids.22 The promising biological functions of POMs have been being widely exploited and applied in clinic diagnosis, i.e., the using of POMs as cheap and efficient therapeutic reagent to diseases23-26 based on the selective linking with specific positive areas of proteins. Great efforts are being taken on about the molecular interaction between POMs and proteins with the aim to further understand the biological functional of POMs.27-29. Fe3O4 nanoparticles (NPs) are a kind of nanomaterial of excellent biocompatibility gaining extensive popularity in the fields of bioimaging, biodiagnosis and therapeutics. The latest studies of Fe3O4 NPs on reducing cytotoxicity, delaying aging and ameliorating neurodegeneration further



demonstrate its great potential in future biomedical applications30-32, and the coating of Fe3O4 NPs with cationic polyethyleneimine (PEI) layer is particularly attractive as it provides the Fe3O4 NPs with specific properties such as improved stability against aggregation33, 34. In this study, the ZrIV substituted POMs (POM1) is firstly prepared and then coated onto the surface of PEI-modified Fe3O4 NPs, giving rise to novel magnetic NPs Fe3O4@PEI@POM1. The preparation strategy not only achieve the solidification of POMs, but also provide the obtained magnetic NPs with negative charge and nanosize. The structure properties of POM1 provide the Fe3O4@PEI@POM1 NPs superior capability on the selective adsorption of IgG via hydrogen-bonding and electrostatic interaction. Selective separation of IgG from human serum is realized with Fe3O4@PEI@POM1 NPs as adsorbent.

EXPERIMENT SECTION Materials and reagents. FeCl3·6H2O, C2H5ONa, trisodium citrate, EG, EtOH, CTAB, SDS, CH3COOH , H3PO4 , HNO3 , H2O2, H3BO3 and NaCl are provided by Sinopharm Chemical Reagent Co., Ltd.(Shanghai, China). 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) and 2morpholinoethanesulfonic acid (MES) are obtained from Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). Immunoglobulin G from human serum (IgG, 14506), bovine serum albumin (BSA, A3311, pI 4.9), transferrin (Trf, T3309, pI 5.25.9), ovalbumin (Ova, A5503, pI 4.7) and branched polyethyleneimine (PEI, Mw=2.5×104 Da) are received from Sigma-Aldrich (St. Louis, USA). The protein molecular weight marker (broad, D532A) is obtained from Takara Biotechnology Company (Dalian, China).


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The above reagents are at lowest of AR grade and used as received. De-ionized water of 18 MΩ cm is adopted during the experimentations. Synthesis of magnetic Fe3O4 NPs. Fe3O4 NPs are obtained according to a reported approach.35 70 mL ethylene glycol and 1.35 g FeCl3·6H2O are added into a 100-mL beaker. After FeCl3·6H2O is dissolved, sodium acetate (3.85 g) and C6H5Na3O7·2H2O (0.4 g) are added and the resultant admixture is heated at 90oC during vigorous stirring until the solution becomes homogeneous claret. Thereafter, the claret solution is transferred into a Teflon-lined stainless-steel autoclave and reacted at 200oC for 16 hours. The black product is then cooled to normal atmospheric temperature. After collection under an external magnetic field, the product is rinsed by EtOH for several times and finally dried at 60oC. Synthesis of polyoxometalate POM1. POM1 is synthesized on the basis of previous work.36 Firstly, 4.94 g H3PW12O40·23H2O is dissolved in 25 mL de-ionized H2O, and the pH of the solution is regulated to 5.25 by adopting 1.0 mol L-1 NaHCO3, then diluted to 50 mL by de-ionized H2O. Thereafter, 0.256 g ZrOCl2 and 1 mL HCl (1.0 mol L-1) are put in to the above solution under vigorous stirring. After stirring for 30 min at normal atmospheric temperature, 0.256 g ZrOCl2 dissolved in 4 mL HCl (1 mol L-1) is added drop-wise and the resultant solution is further stirred for 30 minutes. After filtration, the colorless filtrate is collected and heated at 40°C till the volume becomes 20 mL. Afterwards, the system is heated at 95°C under stirring for 1 min, then 2.5 g Et2NH2Cl is added. After stirring for 5 min, the white suspension is cooled to normal atmospheric temperature and stirred for 1 h. The final product POM1 is gathered by centrifugation, cleaned by EtOH and Et2O successively for several times,



and dried at 40°C in vacuum for 2 hours. Preparation of magnetic Fe3O4@PEI@POM1 NPs. 0.167 g MES, 0.5 g EDC and 0.5 g NHS are dissolved into the 50 mL de-ionized water successively, then 100 mg Fe3O4 is added. After stirring for 30 minutes, the pH of the admixture is regulated to 7 by using NaOH solution. And then PEI solution (500 mg dissolved in 5 mL water) is added under vigorous stirring. After stirring for 3 h, the product is gathered in an external magnetic field. After rinsed by water for several times, the product is redispersed into 50 mL water. Then, 4 mL POM1 (50 mg mL-1) is put in to above suspension and stirred for 12 h. The product is gathered in an external magnetic field, and cleaned with water for some times and finally dried at 60oC. Characterization of Fe3O4@PEI@POM1 NPs. TEM images of the product are achieved by a JEM-1200EX (Hitachi, Japan) at an accelerating voltage of 200 kV. EDXS is obtained by a SU8010 scanning electron microscope (Hitachi, Japan). XRD patterns are taken on a X’Pert Pro MPD X-ray diffractometer (PW3040/60, PANalytical BV, Holland) with Cu Kα radiation (λ=1.5406 Å). FT-IR spectra of Fe3O4@PEI@POM1 are carried out by using Nicolet 6700 spectrometer (Thermo Electron, USA) from 4000 to 400 cm-1. Magnetic characterization is achieved on a vibrating sample magnetometer with a Model 6000 physical property measurement system (Quantum, USA) at 300 K. Zeta potential measurement for surface charge analysis are performed by a Zetasizer Nano ZS90 (Malvern, UK). The adsorption and elution of proteins with Fe3O4@PEI@POM1 NPs. In generally, 2 mg of Fe3O4@PEI@POM1 NPs is put in to1 mL protein sample solution, and then the admixture is shocked on an oscillatore for 10 minutes at normal


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atmospheric temperature to finish the protein adsorption. After protein adsorption is completed, Fe3O4@PEI@POM1 NPs with adsorbed proteins are collected under an external magnetic field. For the recovery of adsorbed protein, Fe3O4@PEI@POM1 NPs are mixed with 1 mL CTAB solution (0.01%, w/v) and shaken for 10 min at normal atmospheric temperature. The solution is thus collected for ensuing investigations after separation in an external magnetic field. The contents of residual proteins in solution after adsorption and the recovered solution are deduced via determining the characteristic absorbance of protein with a U-3900 UV-vis spectrophotometer (Hitachi, Japan). During the adsorption and recovery process, 0.8 mmol L-1 Britton-Robinson (BR) buffer is adopted to adjust the pH values.

RESULTS AND DISCUSSION Synthesis and characteristics of Fe3O4@PEI@POM1 NPs. POMs [Zr(aPW11O39)2]10- is firstly produced by the reaction of [a-PW12O40]3-, with ZrCl2O·8H2O under acid condition, then the sandwich-type POMs, i.e., the 2 (central metals) : 2 (lacunary POMs) type-compounds [{a-PW11O39Zr(µ-OH)(H2O)}2]8- (POM1), is further prepared by the reaction of in-situ generated [Zr(α-PW11O39)2]10- with ZrCl2O·8H2O in HCl solution. The Zr ions in the POMs are able to stabilize the structure of POM1 and avoid gel formation in aqueous solution. Finally, the obtained POM1 is coated on the PEI-modified magnetic NPs via electrostatic interaction between POM1 and PEI to giving to the product of Fe3O4@PEI@POM1 magnetic NPs, as shown in Figure 1.



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Figure 1. The synthetic route of Fe3O4@PEI@POM1 NPs. The TEM images of Fe3O4, Fe3O4@PEI (B) and Fe3O4@PEI@POM1 NPs are illustrated in Figure 2 A-C. The as-prepared Fe3O4 NPs are spherical with average size of 214 nm. After the modification of PEI layer, the size of Fe3O4 increases to 242 nm, and a sheet appears around the Fe3O4 NPs. The coating of PEI modified Fe3O4 NPs with POM1 further causes the increase of Fe3O4 NPs to 263 nm. The size distribution histograms of Fe3O4, Fe3O4@PEI and Fe3O4@PEI@POM1 NPs (Figure 2 D-F) further indicate the gradual increasing of Fe3O4 NPs size with the modifying and coating processes, which is consistent with the results of TEM. A

B

200 nm

200 nm

200 nm

D

200 nm

C

200 nm

200 nm

E

F

Figure 2.TEM images of Fe3O4 (A), Fe3O4@PEI (B) and Fe3O4@PEI@POM1 (C) NPs; Size distribution histogram of Fe3O4 (D), Fe3O4@PEI (E) and Fe3O4@PEI@POM1 (F) NPs.


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Energy dispersive X-ray spectroscopy (EDXS) is implemented to confirm elemental component of these magnetic NPs and the results are summarized in Table 1. The changes on the contents of element in the EDXS of Fe3O4, Fe3O4@PEI and Fe3O4@PEI@POM1 NPs well suggest the successful modification of PEI and coating of POM1 on the surface of Fe3O4 NPs. Table 1. Element contents of Fe3O4, Fe3O4@PEI and Fe3O4@PEI@POM1 NPs. Element contents (wt %) Materials Fe

O

C

N

Zr

P+W

Fe3O4

65.54

31.23

3.23

-

-

-

Fe3O4@PEI

48.84

42.43

7.51

1.22

-

-

Fe3O4@PEI@POM1

32.92

53.39

6.27

0.83

0.19

6.40

The surface charge analysis indicates that the zeta potential of Fe3O4, Fe3O4@PEI and Fe3O4@PEI@POM1 NPs is -18.8 mV, 29.2 mV and -44.3 mV, respectively (Figure 3A), which further confirms the successful fabrication of the magnetic Fe3O4@PEI@POM1 NPs. Figure 3B illustrates the XRD results of the achieved magnetic NPs. The XRD pattern of the Fe3O4 NPs is identical to that of Fe3O4 with the characteristic cubic framework (JCPDS 65-3107). For Fe3O4@PEI and Fe3O4@PEI@POM1 NPs, the 2θ diffraction peaks located at 30.1°, 35.5°, 43.1°, 56.9° and 62.5° correspond to the (220), (311), (400), (511) and (440) planes of Fe3O4, suggesting that the crystal structure of Fe3O4 NPs remains unchanged after PEImodifying and POM1-coating.



Figure 3. Zeta potentials (A), X-ray diffraction (B) of Fe3O4, Fe3O4@PEI and Fe3O4@PEI@POM1 NPs, Fourier-transform infrared spectra (C) of POM1, Fe3O4 Fe3O4@PEI and Fe3O4@PEI@POM1 NPs, and Vibrating sample magnetometer curves (D) of Fe3O4, Fe3O4@PEI and Fe3O4@PEI@POM1 NPs. The peaks at 810, 887, 959, 1057 and 1096 cm-1 appearing in the FT-IR spectra of POM1 (Figure 3C) are corresponded to characteristic bands of Keggin-structure of POMs. In the spectra of Fe3O4 NPs, the stretching vibration of Fe=O at 584 cm-1, the characteristic peaks of symmetric COO-, asymmetric COO- and O-H at 1420, 1607 and 3422 cm-1 are clearly observed. The modification of Fe3O4 NPs with PEI causes the slightly shift of symmetric COO- peak and O-H attributing peak, i.e., from 1420 cm-1 to 1396 cm-1and from 3422 cm-1 to 3404 cm-1, respectively. All the characteristic bands of POM1 (810, 887, 959, 1057 and 1096 cm-1) and Fe3O4 (584, 1420 and 1607 cm-1) appear in the spectra of Fe3O4@PEI@POM1. Additionally, the stretching vibration of O-H shift from 3422 cm-1 to 3518 cm-1 due to the coating of POM1 onto the NPs surface.


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Vibrating sample magnetometer (VSM) is adopted to investigate magnetic properties of the Fe3O4, Fe3O4@PEI and Fe3O4@PEI@POM1 NPs. The saturation magnetization (Ms) value of Fe3O4 NPs is 67.4 emu g-1 (Figure 3D). The PEImodifying and POM1-coating process cause slight reduction on the Ms value, i.e., 63.2 emu g-1 for Fe3O4@PEI and 59.2 emu g-1 for Fe3O4@PEI@POM1 NPs. The VSM results without clear magnetic hysteresis loops, i.e., Hc

Polyoxometalate-Coated Magnetic Nanospheres for Highly Selective

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