pSBMA-conjugated Magnetic Nanoparticles for Selective IgG Separation

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pSBMA-conjugated Magnetic Nanoparticles for Selective IgG Separation Fang Cheng, Chuanlei Zhu, Wei He, Jing Zhao, and Jingping Qu Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00878 • Publication Date (Web): 24 May 2018 Downloaded from http://pubs.acs.org on May 24, 2018

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Langmuir

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pSBMA-conjugated Magnetic Nanoparticles for

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Selective IgG Separation Fang Cheng a ,b,*, Chuanlei Zhu a, c, Wei He a, c, Jing Zhao a, Jingping Qu a

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a

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China

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b

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116030, China

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c

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116030, China

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State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116030,

School of Pharmaceutical Science and Technology, Dalian University of Technology, Dalian

Department of Polymer Science and Engineering, Dalian University of Technology, Dalian

* To whom correspondence should be addressed: [email protected] KEYWORDS: pSBMA, zwitterionic polymer; IgG separation, DVS, nanoparticles, nonfouling

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1 Environment ACS Paragon Plus

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Abstract

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Two types of zwitterionic polymer-modified magnetic nanoparticles (NPs) are fabricated

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by conjugating pSBMA onto PEI-precoated NPs via either one-step method (1S NPs) or two-

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step method (2S NPs). For both methods, divinyl sulfone is used as the linker molecule.

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Although 1S NPs were capable of resisting both IgG and BSA, 2S NPs exhibited specificity

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toward IgG adsorption in complex biological fluids, e.g. mixture of serums and IgG. The

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moderate interactions (Kd ~1.2 µM) between IgG and 2S NPs are three orders of magnitude

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lower than IgG binding with Protein A (Kd 10 nM). Through complementary characterizations

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and analyses, we rationalize that the surface developed herein with IgG specificity contains two

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key components: polyzwitterions with short chain length and sulfone groups with high density.

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Introduction

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Antibody separation is a key biopharmaceutical process, which requires high specificity and

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efficiency in isolating biomacromolecules from a complex biological fluid.1 Development of the

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separation adsorbent benefits diagnostics and therapeutics, such as point-of-care testing2,

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treatment of cancer3 and autoimmune disease4. In the process of antibody separation, Protein A

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chromatography is a commonly employed adsorbent, which could obtain antibody in high purity

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from serum or ascites.5-7 The adsorbent could also eliminate autoantibody as pathogenic from

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patient blood.8 Using the adsorbent immobilized with Protein A, the Fc fragment of

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immunoglobulin is selectively bound in neutral solution and eluted in acidic buffer. In the

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process-scale purification and therapeutic plasma exchange, safety issues, e.g. leakage and

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instability of the immobilized Protein A, and cross-contamination during regeneration, are

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overwhelmed in biopharmaceutics.

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An alternative approach to Protein A chromatography is

the use of synthetic ligand,

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molecular weight of which is commonly less than 200 Da.9 4-Mercaptoethyl pyridine (MEP) is

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such a typical ligand developed for process-scale purification of monoclonal antibody.10-11 In

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biopharmaceutical practice, MEP HyperCel,12 a commercial MEP resin, is applied to separate

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monoclonal antibody from serum-free culture medium. With an optimized ligand density, MEP

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adsorbent has been reported to remove both IgG and IgM family of autoantibodies from patient

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plasma at the same level of Protein A chromatography.13-14 Along with the autoantibodies,

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however, the removal of albumin and other plasma proteins is still challenging.

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The main advantages of synthetic ligands are well-controlled chemical structure, low cost,

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ease in clean-in-place, and repeatable regeneration capability in harsh conditions.9 However, it is


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a challenge to adsorb antibody in a highly selective manner from a complex biological fluid,

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which consists a variety of proteins with a broad range of concentrations. Thiophilic adsorbents,

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bearing structures of sulfone and thioether groups, have been utilized to purify antibody from

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serum or ascites.15-16 Driven by high concentrations of sulfate salts, the adsorbent exhibits

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moderate interactions with disulfide bonds in protein molecule.17 In practice, the precipitation of

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a broad range of proteins on the thiophilic adsorbents leads to nonspecific adsorption in the

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presence of sulfate salts.

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Herein, we report a facile method to develop a quick separation adsorbent, which adsorbs

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antibody from a complex biological fluid with a high specificity. The magnetic nanoparticles

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(NPs) pre-coated with polyethylenimine (PEI) were treated with divinyl sulfone (DVS) and

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subsequently conjugated with thiol-terminated zwitterionic polymer, which was synthesized by

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RAFT polymerization followed by reduction in aqueous solution. The step-wise reactions on the

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magnetic NPs were characterized by XPS and DLS. Using micro-BCA to quantify IgG and BSA,

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the effects of NPs size, polymer molecular weight, solution pH, and salt effect of species and

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concentration on protein adsorption were revealed. Isothermo adsorption and binding affinity

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were investigated using a bio-layer interferometer. The specificity of IgG in simulated complex

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biological fluids, e.g., the mixture of IgG and BSA or mixture of IgG and serum, was examined.

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The scenario of protein adsorption on the adsorbent, i.e. specific adsorption of IgG and resistance

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to other proteins, is proposed.

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Experimental Section

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Reagents and Materials


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[2-(Methacryloyloxy)ethyl]dimethyl(3-sulfopropyl)ammonium hydroxide (SBMA, 97%),

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4,4′-azobis(4-cyanovaleric acid) (v-501, 98%)

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cyanopentanoic acid dithiobenzoate (CADB, 97%）were purchased from Aladdin Industries Co

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Ltd. (Shanghai, China). Divinyl sulfone (DVS, 98%) was obtained from Xiya Chemical Ltd.

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(Shandong,

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piperazineethanesulfonic acid (HEPES) were from J&K (Beijing, China). Sodium chloride,

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sodium sulfate, acetic acid and isopropanol were received from Guangfu Fine Chemical

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Research Institute (Tianjin, China). Bovine serum albumin (BSA, 98%) and human

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Immunoglobulin G (IgG, 95%) were purchased from Melonepharma Technology Ltd. (Dalian,

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China). Fetal bovine serum (FBS) was received from Pan Biotech GmbH. (Aidenbach,

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Germany). Pierce® BCA Protein Assay Kit was purchased from Thermo Scientific (Logan,

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UT). Magnetic nanoparticles with PEI coating were obtained from Nanoeast biotech Co. Ltd.

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(Nanjing, China). A Fc fusion protein, which is composed of an IgG Fc domain that is directly

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linked lipopolysaccharide binding protein, was provided by Prof. Jia Lingyun.

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Preparation of pSBMA and Derivatives (see supporting information Scheme S1)

China).

Sodium

phosphate

sodium borohydride (NaBH4, 98%) and 4-

monobasic

and

4-(2-hydroxyethyl)-1-

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Synthesis of pSBMA: 5,000 mg of SBMA (17.92 mmol), 126 mg of CADB (0.45 mmol)

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and 25 mg of V-501 (0.09 mmol) were dissolved in 25 mL of NaCl solution (0.5 M) and adjusted

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to pH 7.0. The reaction proceeded at 0o C for 3 h under Ar protection, followed by stirring at 70 o

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C for 4 h. The crudes were dialyzed in ultrapure water at 4 o C in dark for 7 days using 3,500 Da

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MWCO membrane. The product (pSBMA, polymer 3) was lyophilized and stored in a

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desiccator.


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Preparation of thiol-terminated polymer (pSBMA-SH): 2,500 mg of pSBMA and 250 mg

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of NaBH4 was dissolved in 10 mL of ultrapure water in an ice bath and the mixture was stirred at

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0 o C for 3 h. The reaction was quenched with 1N HCl, and the pH was adjusted to 3. The

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resulting solution was dialyzed in 20% ethanolic solution, then in ultrapure water at 4°C in dark

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for 4 days. The MWCO of the dialysis membrane was 3,500 Da. The product (pSBMA-SH,

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polymer 1) was lyophilized and stored at -20 o C.

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Preparation of VS-derived polymer (pSBMA-VS): 500 mg of pSBMA-SH and 0.5 mL of

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DVS was dissolved in 10 mL of HEPES buffer (10 mM, pH 9.5, containing 10% acetonitrile,

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v/v) and the solution was stirred at 25 o C for 6 h. The resulting solution was dialyzed in 20%

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ethanol, then in water at 4°C in dark for 5 days, using 3,500 MWCO membrane. The product

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(pSBMA-VS) was lyophilized and stored at -20 o C.

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Proton NMR: 1H NMR spectra were acquired using a Bruker Avance II-400 MHz

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spectrometer with D2O as the solvent. At least 64 scans were recorded for each sample. All

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chemical shifts (δ) are reported in ppm. (see supporting information Figure S1 for pSBMA and

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derivatives)

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GPC analysis: Gel permeation chromatography (GPC) was performed with a Waters

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Breeze 2 GPC instrument equipped with three columns (UltrahydrogelTM 120, 250 and 500) and

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a Waters 2414 refraction index detector (Waters Corporation, USA). Briefly, 20 µl of the

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polymer sample (0.5 wt% in water) was injected and eluted with 0.1 M NaNO3 at 0.8 ml/min.

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The column temperature was controlled at 35.0 ± 0.2 o C and the results were recorded by on-line

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computer. The number average molecular weight was calculated according to the calibration

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curve generated using poly(oxyethylene) standards.


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Preparation of pSBMA-conjugated NPs

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To render the NPs the capability of protein resistance, we developed two approaches

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using pSBMA derivatives as starting materials (Scheme 1). First, zwitterionic monomer (SBMA)

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was polymerized in degassed aqueous solution using RAFT technique. Sequentially,

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thiocarbonythio groups of the freshly prepared pSBMA were converted into thiol groups using

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strong reduction reagents, e.g., sodium borohydride. The resulting pSBMA-SH was treated with

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an excess amount of divinyl sulfone (DVS) to obtain pSBMA-VS. (see supporting information

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Scheme S1). Taking advantages of the versatile reactivity of VS group toward amines, the

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purified pSBMA-VS was conjugated onto the PEI-precoated NPs via a one-step method (Scheme

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1a). The resulting NPs are referred to as 1S NPs. Alternatively, a two-step method was

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developed to conjugate purified pSBMA-SH onto the DVS-treated NPs via Scheme 1b, resulting

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in 2S NPs. Experimental details of the preparation of pSBMA-conjugated NPs are as follows.

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Preparation of 1S NPs by a one-step method: 1 mg of magnetic NPs pre-coated with PEI

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was dispersed in 2.0 mL of pSBMA-VS solution (15 mg/mL in 10 mM HEPES buffer, pH 9.5)

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and the mixture was incubated for 10 h at 25 °C. Then, the resulting 1S NPs were collected by

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centrifugation at 13,500 rpm for 15 min and purified through triplet washes with 1 mL of water.

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Preparation of 2S NPs by a two-step method：1 mg of magnetic NPs pre-coated with PEI

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was dispersed in 0.8 mL of HEPES buffer (10 mM, pH 9.5). 1.2 mL of DVS solution (30% in

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acetonitrile, v/v) was added and the mixture was stirred for 10 h at 25°C. Then, the DVS-

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treated NPs were collected by centrifugation at 13,500 rpm for 15 min and purified through

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triplet washes with 1 mL of ethanol. Next, 1 mg of the DVS-treated NPs was dispersed in 2.0 mL

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of pSBMA-SH solution (15 mg/mL in 10 mM phosphate buffer, pH 7.4) and the mixture was


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incubated for 4 h at 25 °C. Then, the resulting 2S NPs were collected by centrifugation at

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13,500 rpm for 15 min and purified through triplet washes with 1 mL of water.

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XPS Characterization

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XPS measurements were conducted on a Thermo Scientific ESCALAB 250Xi X-ray

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photoelectron spectrometer (Waltham, MA). All XPS data were acquired at a photoelectron

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takeoff angle of 90°. Thermo advances and XPS peak software were used to calculate elemental

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compositions and fit high-resolution spectra, respectively.

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DLS Analysis

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The hydrodynamic diameter was measured using dynamic light scattering technique on a

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Brookhaven 90 plus PALS particle size analyzer. Samples were prepared by dispersing NPs in

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ultrapure water using ultrasonicator at a final concentration of 0.1 mg/mL. The hydrodynamic

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diameter was reported as a mean of at least 5 measurements.

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Protein Adsorption Assessment

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1 mg of NPs was dispersed in 1 mL protein solution (1 mg/mL in phosphate buffer, pH

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7.4) and the mixture was incubated at room temperature for 10 min. A rocking platform was used

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to provide gentle mixing. Then, the NPs were separated with a magnet and purged through triplet

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washes with 0.2 mL of phosphate buffer. The washing buffer was mixed with the protein

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solutions extracted by the NPs, and the protein concentration was measured using Pierce® BCA

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Protein Assay Kit. The amount of protein adsorption was determined by the difference between

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original protein concentration and residual protein concentration. For all experiments, triplicate


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samples were included. Statistical analysis was performed using two-tailed Student's t-test (two-

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group comparison).

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Bio-layer Interferometer

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A BLItz biolayer interferometer (Pall Fortebio, U. S. A.) was used for the measurements of

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protein adsorption isotherm and kinetics. An amine-modified sensor probe was conjugated with

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pSBMA using the two-step method. For protein adsorption measurements, the following

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protocol was used: the biosensor was firstly immersed in running buffer for 30 s, then dipped in a

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buffered protein solution for 120 s, followed by immersion in running buffer for 120 s. The

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probe was regenerated using 40 mM acetic buffer (pH 4) for 120 s, and washed by running

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buffer for 60 s. The running buffer used in all the experiments was 10 mM phosphate buffer (pH

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7.4) unless otherwise stated. Running buffers (pH 7.4) containing different concentrations of

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proteins (0.01−3.0 mg/mL) were used for the protein adsorption measurements. The adsorption isotherm was fitted to the Langmuir equation18,

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 =

+ eq. 1

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

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where and are the BLItz response to equilibrium adsorption and the equilibrium protein

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concentration, respectively. is the maximum binding capacity, and is the dissociation

168

constant.

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Results and Discussion

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Initial experiments of protein adsorption on NPs


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A pilot study of protein adsorption on pristine NPs, 1S NPs and 2S NPs was carried out

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using IgG and BSA. As shown in Figure 1, IgG and BSA barely adsorbed on 1S NPs, 19-20 while

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both proteins adsorbed readily on the pristine NPs because of the positively charged PEI coating.

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Comparing with the nonfouling 1S NPs, we observed a significant increase in IgG adsorption on

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2S NPs, and a drastic decrease in terms of BSA adsorption. To confirm the high specificity, the

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selective adsorption of IgG in simulated biological fluids, e.g., the mixture of IgG and serum or

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mixture of IgG and BSA, was analyzed by SDS-PAGE (see supporting information Figure S2).

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In light of these results, we then focused on the 2S NPs, characterizing the step-wise surface

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reaction and protein adsorption behaviors, and revealing the mechanism of selective adsorption

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of IgG.

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Characterization of pSBMA-conjugated NPs by XPS and DLS

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The step-wise surface reaction for preparing 2S NPs was characterized by XPS. The

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elemental compositions were examined by survey scans (Figure 2A). The organic compositions

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of the NPs are summarized in Table 1. High intensities of Fe2p and N1s are observed on the

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pristine NPs, likely due to pre-coated PEI on the pristine NPs with Fe3O4 core. After treated with

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DVS, an increase in S2p is assigned to the sulfone groups covalently bound on the surface.21-22

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Once conjugated with the thiol-terminated pSBMA (pSBMA-SH), the ratio of N1s to S2p is

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approximately 1.2, which is close to the stoichiometric ratio in pSBMA.23 For comparison

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purpose, as-prepared 1S NPs was also characterized. Although pSBMA-VS conjugated on the

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surface of 1S NPs, the ratio of N1s to S2p is approximately 2.0. Because of partial attenuation of

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the signals of N1s stemming from the PEI-precoating, a sparse layer of pSBMA was prepared on

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1S NPs.


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The high-resolution spectra of S2p (Figure 2B), C1s and O1s (Figure S3) are also collected.

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For the pristine NPs, traces of sulfur species are detected on the surface. After treated with DVS,

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a doublet peak around 168 eV is detected and can be assigned to oxidized sulfur species

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originating from the sulfone groups.21, 24 Once conjugated with pSBMA-SH, in addition to the

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peak at 168.0 eV, another doublet peak around 162 eV appears, corresponding to reduced sulfur

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species in the resulting thioether groups on 2S NPs. The peak at 168.0 eV, which is indicative of

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oxidized sulfur species,25 can be attributed in part to the sulfonate groups in pSBMA. Similar to

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2S NPs, both oxidized and reduced sulfur species are detected on 1S NPs surface. The ratio of

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the oxidized species to the reduced species is slightly lower than that of 2S NPs, which can be

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explained by the high concentration of surface sulfone groups after DVS treatment.

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Change in particle size is a good index to gauge the success in surface modification. The

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hydrodynamic diameter (Dh) of NPs in water was examined using DLS, where Dh was calculated

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to be a number weighted average. The DLS measurements are given in Table S1. Compared with

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pristine NPs, significant changes in Dh for both 2S NPs and 1S NPs are observed. Particularly,

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average Dh of 2S NPs is approximately 40% larger than that of 1S NPs. In good agreement with

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the above XPS characterization, it suggests that the two-step method results in a dense layer of

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pSBMA on 2S NPs.

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Effects of NPs size and polymer molecular weight on protein adsorption behavior

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The two-step method has been applied to three types of magnetic NPs with different sizes,

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all pre-coated with PEI. Figure 3A shows the amount of IgG and BSA adsorbed on the three

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types of 2S NPs. For IgG, the amount of adsorption decreases as the size of NPs decreases.

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Whereas the BSA adsorption shows an opposite trend. Using the specificity parameter defined as


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the ratio of the amount of adsorbed IgG to that of adsorbed BSA, the size-dependent specificity

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is calculated to be 1.6, 3.8 and 28.0 for 25 nm, 50 nm and 1 µm particles, respectively. It is worth

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noting that the total mass of NPs used in these experiments was kept the same. The results

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suggest that the large size NPs of a small number have high specificity, while the small size NPs

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of a large number have good capability of protein resistance.

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Three types of pSBMA with different molecular weights (see Figure S4 for GPC analysis)

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were conjugated onto DVS-treated NPs using the two-step method. The amount of IgG and BSA

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adsorbed on the three types of 2S NPs is shown in Figure 3B. For 2S NPs with short pSBMA

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chains, i.e., Mn 4389 and 7611, the amount of IgG adsorption is significantly higher than that of

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2S NPs with long chains. It could be due to short pSBMA chains on the surface exposing more

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sites for IgG specific binding. Yet, the short pSBMA chain on the surface provides effective

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resistance to BSA. Once conjugated with long pSBMA chains, the capacity of IgG binding is

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lower, while surface resistance to BSA is not sufficient. It has been reported that surface

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resistance to protein adsorption is dependent on the film thickness of polyzwitterion. In general,

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with the increase in the polymer film thickness, the capability of the resistance to protein

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increases remarkably. However, further increase of the film thickness leads to some level of

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protein nonspecific adsorption, because of the possible conformational self-condense of long

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polymer chains.26 These results were obtained on the polymer film prepared by bottom-up

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methods, e.g. surface-initiated ATRP.27-28 Although our 2S NPs are prepared by a top-down

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method, similar trends are observed due to the intrinsic nature of the polymer chain.

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Effects of solution pH on protein adsorption behavior


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Langmuir

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The solution pH was a significant factor for both IgG and BSA adsorption. As can be

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seen in Figure 4A, the maximum amount of IgG and BSA adsorption occurs in pH 6-7 and pH 4,

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respectively. It is consistent with the fact that protein tends to adsorb on surface at its isoelectric

239

point.10 Interestingly, the amount of IgG adsorption decreases drastically in acidic buffer

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solution. Although pSBMA is neutral in a broad range of pH, both IgG molecules and PEI-

241

coated NPs carry positive charges in acidic solution. The electrostatic repulsion leads to decrease

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in protein adsorption. Therefore, acidic buffer could be adopted to remove the adsorbed IgG on

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2S NPs. Even after five times of acidic regeneration, 2S NPs exhibit good capability of IgG

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binding and specificity (Figure 4B).

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Effects of salt species and concentration on protein adsorption behavior

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Salt concentration and species affect the protein adsorption behaviors differently. The

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amount IgG and BSA adsorption on 2S NPs is shown in Figure 5. For BSA, both sodium

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chloride and sodium sulfate slightly induced an increase in adsorption in high concentration of

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salt (0.6 M). For IgG, sodium chloride and sodium sulfate affected protein adsorption differently

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in a broad range of salt concentrations (0 – 0.6 M). It can be explained by the fact that chain

251

conformations of polyzwitterions and polyelectrolytes exhibit contrasting behaviors under the

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influence of Hofmeister salt series.29 Delgado et al. described a consistent scenario of pSBMA

253

conformation, which illustrated four or more regimes of pSBMA conformation/interaction as a

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function of ionic strength.29 Because isoelectric points of IgG and BSA are ~6-7 and 4.6, the

255

protein molecules could be considered as polyzwitterions and polyelectrolytes in neutral buffer

256

solution, respectively.

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Isothermo adsorption and binding affinity


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Protein adsorption behaviors of IgG and BSA were examined using a bio-layer

259

interferometer. The amine-coated surface of interferometer probe was conjugated with pSBMA

260

using the two-step method. As shown in Figure 6A, IgG adsorption with high specificity was

261

supported by the real-time sensorgrams, which was consistent with the above results obtained on

262

2S NPs. The adsorption isotherms of IgG on 2S surface were determined. The concentration-

263

dependent data (Figure 6B) were fitted with the Langmuir equation (eq. 1), the dissociation

264

constant (Kd) was calculated to be 0.18 mg/mL (1.2 µM). The typical dissociation constant of

265

IgG bound with Protein A is 10 nM. The drastic decrease in the affinity leads to moderate

266

interactions between IgG and 2S surface. To clarify the interactions, the adsorption behaviors of

267

an Fc fusion protein on 2S surface were also examined. Interestingly, the adsorption of the fusion

268

protein was not detected (Figure 6A). In general, Fab domains of IgG contain disulfide bonds,

269

which lead to thiophilic interactions with the sulfone and thioether groups. Compared with the

270

control, the selective adsorption of IgG evidenced by both 2S NPs and biosensor suggests that

271

the interactions between 2S surface and IgG would be thiophilic.

272

Scenario of protein adsorption behavior on 2S NPs

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In this work, we observed that the selective adsorption of IgG on 2S NPs, while 1S NPs

274

exhibit good resistance to both IgG and BSA. Compared with 1S NPs, 2S NPs prepared by the

275

two-step method presents more sulfone groups on the 2S surface. We propose that IgG

276

adsorption is predominantly determined by the dense sulfone groups, while the contribution of

277

sulfone-thioether structures for IgG adsorption is negligible. Therefore, the surface exhibiting

278

good specificity for IgG adsorption would contain two key components as follows:

279

polyzwitterion with short chain length and sulfone groups with high density. The schematic

280

diagram of protein adsorption on 2S NPs is proposed in Figure 7. In neutral conditions, the


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dominant interactions between dense sulfone groups and IgG molecules lead to IgG adsorption,

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while short chain pSBMA resists other proteins. In acidic buffer, the electrostatic repulsions

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mainly cause the removal of positively charge IgG molecules from PEI pre-coatings.

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In general, magnetic nanoparticles displayed with synthetic ligands have been reported to

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adsorb IgG antibody in a selective manner.30-33 Typically, the binding capacity can reach 140-

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170 mg IgG per gram of support, while the binding affinity is estimated in the range of

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micromolar.30-32 Multiple steps of surface treatments are involved in the preparation of such

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magnetic nanoparticles. Initially, a layer of natural polymer, e.g., dextran or extracellular

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polysaccharide, is coated onto the bare nanoparticles for protein resistance. Subsequently, the

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coated polymers are activated and conjugated with synthetic ligands.30-33 The activation methods,

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which in turn determine spacing arm and ligand density on the nanoparticles, directly impact the

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binding capacity and affinity. For the 2S method developed herein, thiophilic groups followed by

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antifouling polyzwitterions were introduced on the nanoparticles. Because of the friendly RAFT

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polymerization in aqueous solution, the length of grafted polymers is tunable with a narrow

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polydispersity index. Moreover, a library of zwitterionic monomers34 could be explored,

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providing more options for surface functionalization of magnetic nanoparticles.

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Conclusions

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In this work, a facile method to prepare an adsorbent for IgG separation was developed.

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The adsorbent (2S NPs) exhibits IgG adsorption with high specificity in simulated complex

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biological fluids, and could be used repeatedly at least five times. A variety of factors pertaining

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to NPs and solutions, including NPs size, polymer molecular weight, solution pH, salt species

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and concentration, and regeneration, are examined. Among them, two key components


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presenting on the NPs surface, i.e. short pSBMA chain and dense sulfone groups, are essential.

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These findings could facilitate the preparation of new adsorbents for antibody separation and

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purification.

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Scheme 1. Preparation of pSBMA-conjugated NPs: (a) 1S NPs via a one-step reaction using

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pSBMA-VS, (b) 2S NPs via a two-step reaction using pSBMA-SH.

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Figure 1. Amount of IgG and BSA adsorption on NPs: (a) pristine NPs, (b) 1S NPs, and (c) 2S

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NPs conjugated with pSBMA. Data are presented as the mean ±SD, n = 3. (NS) No significance

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and (***) p < 0.001 compared with the IgG adsorption.

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Figure 2. XPS characterization: (A) survey scans and (B) sulfur high-resolution analysis for (a)

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pristine NPs, (b) DVS-treated NPs, (c) 2S NPs and (d) 1S NPs.

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Figure 3. Effects of (A) NPs size and (B) pSBMA molecular weight on protein adsorption

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behavior for 2S NPs. Data are presented as the mean ±SD, n = 3. (NS) No significance, (**) p

pSBMA-conjugated Magnetic Nanoparticles for Selective IgG Separation

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