Label-Free Specific Detection and Collection of C-Reactive Protein

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Label-free specific detection and collection of C-reactive protein using zwitterionic phosphorylcholine-polymer-protected magnetic nanoparticles Sana Iwasaki, Hideya Kawasaki, and Yasuhiko Iwasaki Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01007 • Publication Date (Web): 04 May 2018 Downloaded from http://pubs.acs.org on May 5, 2018

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Langmuir S. Iwasaki et al.,

1

Label-free specific detection and collection of C-reactive protein using zwitterionic phosphorylcholine-polymer-protected magnetic nanoparticles

Sana IWASAKI, Hideya KAWASAKI and Yasuhiko IWASAKI*

Department of Chemistry and Materials Engineering; Faculty of Chemistry, Materials and Bioengineering, Kansai University, 3-3-35 Yamate-cho, Suita-shi, Osaka 564-8680; Japan

Fax: +81-6-6368-0090; Telephone: +81-6-6368-0090 E-mail: [email protected]

*Corresponding author

ACS Paragon Plus Environment

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Page 2 of 34 S. Iwasaki et al.,

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ABSTRACT In

this

study,

poly[2-methacryloyloxyethyl

phosphorylcholine

(MPC)]-protected Fe3O4 nanoparticles were prepared and used for the label-free specific detection and collection of an acute inflammation marker, C-reactive protein (CRP), in a simulated body fluid. The Fe3O4 nanoparticle surface was modified using poly(MPC) by surface-initiated atom transfer radical polymerization. The density of poly(MPC) was 0.16 chains/nm2, and the colloidal stability of the nanoparticles in aqueous media and human plasma was effectively improved by surface modification. The size of the as-prepared poly(MPC)-protected Fe3O4 nanoparticles was ~200 nm. After coming into contact with CRP, the nanoparticles aggregated as CRP comprises five subunits, and each subunit can bind to a phosphorylcholine group with two free Ca2+ ions. The change in the nanoparticle size exhibited a good correlation with the CRP concentration in the range of 0–600 nM. A low limit of detection of 10 nM for CRP was observed. The particles effectively reduced the adsorption of nonspecific proteins, and the change in the nanoparticle size with CRP was not affected by the coexistence of bovine serum albumin at a concentration 1000 times greater than that of CRP.

Nanoparticle

aggregates

formed

ethylenediamine-N,N,N′,N′-tetraacetic acid,

using

CRP

disodium

were

dissociated

using

salt, thereby regenerating

poly(MPC)-protected Fe3O4 nanoparticles. In addition, CRP was collected from aqueous media using an acidic buffer solution and human plasma. CRP-containing aqueous solutions were treated with poly(MPC)-protected Fe3O4. After separating poly(MPC)-protected Fe3O4 using a neodymium magnet and centrifugation, the concentration of CRP in the media dramatically decreased. In stark contrast, the concentration of albumin present in the


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test solution did not change even after treatment with the nanoparticles. Therefore, nanoparticles specifically recognize CRP from complex biological fluids. Although inhibition tests with the presence of 1,2-dioleoyl-sn-glycero-3-phosphocholine liposomes or free poly(MPC) were also carried out, the binding of poly(MPC)-protected Fe3O4

to

CRP

was

not

affected

by

these

inhibitors.

In

conclusion,

poly(MPC)-brush-bearing magnetic nanoparticles can serve not only as reliable materials for detecting and controlling the levels of CRP in simulated body fluids but also as diagnostic and therapeutic materials.

KEYWORDS 2-methacryloyloxyethyl phosphorylcholine / magnetic nanoparticle / polymer brush / ATRP / C-reactive protein (CRP) / biosensor / inflammation

INTRODUCTION The exploration of a rapid, simple, and reliable method for detecting

inflammation markers is interesting for preventive and therapeutic applications.1 C-reactive protein (CRP) is well known to be one of the objective makers for inflammation because the concentration of CRP in serum increases by >3000-fold in response to infection, tissue injury, or other inflammatory conditions.2 Furthermore, CRP is an important risk factor for coronary cardiovascular disease.3-5 CRP is known to be present in atherosclerotic plaques and induces the expression of chemokine and adhesion molecules in endothelial cells.6 Hence, it is crucial to detect and control the CRP level in patients for therapeutic treatment. Currently, turbidimetric immunoassays are conventionally used to quantify the CRP concentration. Although these


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immunoassays constitute a well-established, reliable method, antibodies are required, and this method is not applied for the elimination of CRP from body fluids. Meanwhile, magnetic nanoparticles have been widely used as attractive tools for biomedical purposes such as magnetic resonance imaging, separation, hyperthermia, biosensing, and bioanalysis.7-9 The use of hydrophilic molecules, including water-soluble polymers, for controlling the surface properties of magnetic nanoparticles is crucial for the application of magnetic nanoparticles in biomedical applications.10,11 Biochemical separation is one of the most significant applications of magnetic nanoparticles. Herrmann et al. have summarized recent studies on the removal of specific compounds from whole blood using magnetic nanoparticles.12,13 The removal system works well, but it is imperative to use a capture agent that can bind to a specific molecule. Biomolecules such as peptides, proteins, nucleic acids, and carbohydrates have been used as capture agents, which are powerful tools for the specific recognition of target molecules. However, the immobilization of biomolecules on solid surfaces can lead to their denaturation and inactivation.14 Furthermore, the low stability of biomolecules in body fluids may pose an issue. Sometimes capture agents limit the applicability of magnetic blood purification. In this study, the label-free detection and collection of CRP using biomimetic magnetic nanoparticles was proposed. CRP is synthesized in the liver. The native form of CRP is “pentraxin” (115 kDa), which comprises five identical subunits (23 kDa) with noncovalent protomers arranged in a symmetrical pentagon around a central pore.15,16 Each protomer has a phosphorylcholine (PC) binding site with two coordinated Ca2+ adjacent to a hydrophobic pocket. Phenylalanine (Phe)-66 and glutamic acid (Glu)-81 are the two key residues that mediate the binding of PC to CRP.17 Specific binding


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between various synthetic phospholipid analogs and CRP has been reported because CRP is

related

to

the

biology

of

various

diseases.5,

18-20

In

particular,

2-methacryloyloxyethyl phosphorylcholine (MPC) is one important candidate for this purpose.21 Goda et al. first reported the label-free detection of CRP with MPC copolymers.22 To quantify the activation dynamics of human CRP, the authors synthesized to detect bearing active esters and immobilized the copolymers on amino functional substrate for SPR. The binding of CRP to the PC receptor is dependent on the local [Ca2+] and pH. In addition, the CRP activation on the MPC copolymer surface was monitored using an organic bioelectronic ion pump system. In contrast, our group has prepared MPC-block-copolymer-bearing gold nanoparticles.23 Excellent colloidal stability of gold nanoparticles is observed at high salt concentrations and over a wide pH range. When CRP makes contact with MPC-copolymer-immobilized gold nanoparticles, nanoparticles spontaneously aggregate, which precipitate at a CRP concentration of greater than 40 nM. Poly(MPC)-protected Fe3O4 nanoparticles in this study exhibited excellent colloidal stability in aqueous media. The size of the nanoparticles changed during their contact with CRP and showed a good correlation with the CRP concentration. Moreover, CRP was selectively collected from diluted serum. Hence, poly(MPC) brush-bearing magnetic nanoparticles can be reliable materials for detecting and controlling the CRP levels in simulated body fluids as well as diagnostic and therapeutic materials.

MATERIALS AND METHOD

Materials 3-(2-Bromoisobutyryl)propyl dimethylchlorosilane (BDCS) was synthesized


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according to a previously reported method.24 Purified water (reverse osmosis) was further purified using a Millipore Milli-Q system with reverse osmosis, ion exchange, and filtration steps (18.2 MΩ). MPC and phospholipids were donated by NOF Co., Tokyo, Japan. CRP was purchased from Sigma-Aldrich Inc., St Louis, MO, USA. Human serum was obtained from Biowest, Nuaillé, France. Other chemicals obtained from Wako Pure Chemical Industries, Ltd., Tokyo, Japan, were used without further purification.

Synthesis of poly(MPC)-protected magnetic nanoparticles Figure 1 shows the schematic for the preparation of poly(MPC)-protected Fe3O4 nanoparticles. Fe3O4 nanoparticles were prepared by hydrothermal synthesis. First, FeCl2·4H2O (1.88 g, 9.46 mmol) was dissolved in 10.5 mL of water and 10.5 mL of 28% ammonium hydroxide. The suspension was stirred for 1 h at 250 rpm. Next, the reaction mixture was heated in a sealed pressure vessel (TAF-SR, Taiatsu Techno Co., Tokyo, Japan) at 135 °C for 3 h and cooled to room temperature. The black precipitate was rinsed with water (15 mL × 2) and methanol (15 mL × 3) by magnetic separation. The final Fe3O4 nanoparticle suspension was dried under reduced pressure, affording a dry black powder. The yield of Fe3O4 nanoparticles was 39.7 %. First, Fe3O4 nanoparticles (0.5 g) were placed in a dry scintillation vial, and 10 mL of dry toluene was added into the vial, followed by ultrasonication to achieve a better dispersion. Then, 2.5 mL of toluene was added into a 100-mL three-necked round-bottom flask containing the Fe3O4 nanoparticle suspension. BDCS (1 mL, 4.54 mmol) was added into the suspension under argon. The flask was subjected to sonication and allowed to stand for 24 h. The nanoparticles were rinsed with toluene


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and acetone, followed by drying under reduced pressure. A mixed solvent comprising one portion of acetone and one portion of water was used for the ATRP of MPC.24 Argon was purged in these solvents to eliminate oxygen before polymerization. Copper(I) bromide (29 mg, 0.20 mmol) and 2,2′-dipyridyl (63 mg, 0.40 mmol) were dissolved in 4 mL of solvent for ATRP with stirring under argon. Then, ethyl 2-bromoisobutyrate (9 µL, 0.060 mmol) was added as the sacrificial initiator. The BDCS-immobilized Fe3O4 nanoparticles (100 mg) were placed in a dry vial, and 5 mL of the solvent for ATRP was added, followed by ultrasonication to achieve better dispersion and purging with argon for 60 min to eliminate oxygen. After the suspension was stirred for 10 min under argon, the BDCS-immobilized Fe3O4 nanoparticle suspension was poured into the flask. The empty vial was rinsed using 7 mL of the mixed solvent, and the rinsing solution was added into the flask. First, MPC (12 g, 0.041 mol) was separately dissolved in 14 mL of the mixed solvent purged with argon for 60 min to eliminate oxygen. The MPC solution was added to the flask, and polymerization was carried out at room temperature with stirring under argon for 4.5 h. Poly(MPC)-protected Fe3O4 nanoparticles were periodically removed from the polymerization mixture, rinsed using water by centrifugation– dispersion, and freeze-dried.

Characterization of poly(MPC)-protected magnetic nanoparticles Transmission electron microscopy (TEM). TEM images were recorded on a JEOL model JEM-1400 instrument operated at an accelerating voltage of 100 kV. A sample was prepared by placing a drop of a nanoparticle solution on a collodion-coated


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copper grid, followed by air-drying. Dynamic light scattering (DLS) analysis. A Malvern Zetasizer Nano-ZS system operating at a laser wavelength of 633 nm and a fixed detector angle of 173° was utilized for DLS measurements using highly dilute aqueous dispersions. Thermogravimetric analysis (TGA). TGA curves were recorded using a Rigaku Thermo plus TG-8120 instrument. Samples were heated from 25 °C to 530 °C at 5 °C/min. The surface density of poly(MPC) (chains/nm2) was estimated by the following equation (1)8: % 4 % 3 = , (1) 4 where wt%shell is the relative mass of the polymer, wt%core is the residual mass of the Fe3O4 nanoparticles, ρcore is the density of the Fe3O4 nanoparticles (ρcore = 5.17 g/cm3), 4/3πrcore3 is the volume of a single particle, NA is the Avogadro’s number, Mn is the number-average molecular weight of the polymer, and 4πrcore2 is the surface area per particle.

Interaction of C-reactive protein (CRP) with poly(MPC)-protected magnetic nanoparticles Poly(MPC)-protected

magnetic

nanoparticles

were

suspended

in

2-morpholinoethanesulfonic acid monohydrate (MES, pH 5.5), and the concentration was adjusted to 0.5 mg/mL. The suspension (15 µL) was mixed with 500 µL of 2 mM CaCl2 in an MES buffer in a plastic tube. Appropriate amounts of the CRP/MES buffer at a concentration of 1826 nM were then added to the tube. The total volume of the test liquids was adjusted to 1.0 mL. After mixing all samples, the tubes were stored at room


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temperature for 60 min, and the nanoparticles were precipitated using a neodymium magnet, followed by redispersion by pipetting 7 times and DLS measurements for size analysis and TEM observation. In addition, the effect of the presence of 3.5 mg/mL of bovine serum albumin (BSA) on the interaction between nanoparticles and CRP was determined. In addition, poly(MPC)-protected magnetic nanoparticles with CRP were regenerated using 10 mM of ethylenediamine-N,N,N′,N′-tetraacetic acid (EDTA). CRP was kept in contact with poly(MPC)-protected magnetic nanoparticles under similar conditions mentioned above. The final concentrations of CRP and nanoparticles were adjusted to be 30 nM and 7.5 µg/mL, respectively. After DLS measurements, 110 mM of EDTA (100 µL) was added into the suspension (1.0 mL) containing aggregates of poly(MPC)-protected magnetic nanoparticles with CRP. Then, the suspension was gently mixed and stored for 30 min. The nanoparticle size was measured again by DLS. After rinsing the nanoparticles three times with the MES buffer, the nanoparticles were then repeatedly placed in contact with the same CRP concentration in the presence of 1 mM CaCl2.

Collection of C-reactive protein (CRP) using poly(MPC)-protected magnetic nanoparticles Poly(MPC)-protected magnetic nanoparticles were dissolved in 0.1 M MES buffer, and the concentration was adjusted to 0.5 mg/mL. The suspension (200 µL) was mixed with 500 µL of 2 mM CaCl2 in the MES buffer in a plastic tube. Appropriate amounts of the 1826 nM CRP/MES buffer were then added to the tube. The total volume of the test liquids was adjusted to 1.0 mL.


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Page 10 of 34 S. Iwasaki et al., 10

After 2-h incubation, the nanoparticles were precipitated using a neodymium magnet. The supernatant (500 µL) was collected and further subjected to centrifugation twice at 10,000 rpm for 40 min (PMC-060, TOMY SEIKO Co., Ltd., Tokyo, Japan) to precipitate the nanoparticles. The concentration of CRP in the supernatant (250 µL) was determined using an ELISA kit (Immunology Consultant Lab Inc., Portland, USA). The adsorbed amount of BSA on nanoparticles was also determined by the same procedure using the ELISA kit (Bethyl Laboratories, Inc., Montgomery, USA). In addition, inhibition tests for the binding of poly(MPC)-protected Fe3O4 nanoparticles

and

CRP

were

carried

out

in

the

presence

of

1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) liposome or free poly(MPC) as the inhibitor. For this test, the concentration of CRP in the MES buffer was first adjusted to 120 nM. Poly(MPC)-protected Fe3O4 nanoparticles (0.5 mg/mL) were added into the solution with or without inhibitors and incubated under similar condition as mentioned above. The calculated ratio of the PC groups of poly(MPC) to the CRP sub units was 113. During the inhibition test, the composition of the PC groups of inhibitors was equal to that of the nanoparticles.

RESULTS AND DISCUSSION Armes et al. were the first to prepare poly(MPC)-protected Fe3O4

nanoparticles.25

A

double-hydrophilic

poly[MPC-block-(glycerol

monomethacrylate)]

diblock was

copolymer synthesized,

comprising and

magnetic

nanoparticles were prepared by the coprecipitation of ferric and ferrous salts in the presence of the copolymer with the addition of ammonium hydroxide. The colloidal stability of nanoparticles was well improved by surface modification at pH ranging


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Langmuir S. Iwasaki et al., 11

from 1 to 12, and poly(MPC) block appeared to confer enhanced colloidal stability compared to the PEG block. Furthermore, the surface of magnetic nanoparticles with MPC polymers was modified by surface polymerization methods.26,27 Although the characterization and improved colloidal stability of Fe3O4 nanoparticles have been reported previously, biointerfacial aspects, i.e., nonspecific interaction with plasma proteins

and

specific

molecular

recognition

on

poly(MPC)-protected

Fe3O4

nanoparticles, have not been well investigated. In this study, a specific protein was specifically detected and separated using poly(MPC)-protected Fe3O4 nanoparticles. To generate dense poly(MPC) brushes on Fe3O4 nanoparticles, the SI-ATRP of MPC was carried out. Figure 2 shows the FTIR spectra of bare Fe3O4 nanoparticles, BDCS-immobilized Fe3O4 nanoparticles, and poly(MPC)-protected Fe3O4 nanoparticles. A peak located at 576 cm−1 corresponding to the Fe–O stretching band was considerably observed in the spectrum of bare Fe3O4 nanoparticles. An alternative peak corresponding to the Si–O stretching vibration, related to the immobilization of BDCS on Fe3O4 nanoparticles, was observed in the spectrum of BDCS-immobilized Fe3O4 nanoparticles. The Fe–O peak diminished in the spectrum of poly(MPC)-protected Fe3O4 nanoparticles, and peaks corresponding to phosphoester were observed. Peak characteristics of phosphodiesters were observed. P=O stretching and asymmetric P–O–C stretching vibrations were observed at ~1160 and ~970 cm−1, respectively, indicative of the successful surface immobilization of poly(MPC) on Fe3O4 nanoparticles. Figure 3 shows the size distribution (by intensity) of the bare Fe3O4 nanoparticles and poly(MPC)-protected Fe3O4 nanoparticles. The average particle was 165.4 nm, with a PDI of 0.21 according to the DLS results, indicative of a


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well-monodispersed solution. As a result of poly(MPC) immobilization, the nanoparticle diameter increased. The averaged diameter of the poly(MPC)-protected Fe3O4 nanoparticles became 258.8 nm (PDI = 0.106). The molecular weight of the poly(MPC) brushes generated on Fe3O4 nanoparticles was determined by the GPC analysis of free poly(MPC) formed by the additional

sacrificial

initiator.

The

number-average

molecular

weight

of

poly(MPC)-protected on Fe3O4 nanoparticles was estimated as 1.05 × 105 (DP ≈ 350), and the molecular weight distribution was 1.03. The hydration layer of poly(MPC) can be estimated at approximately 60 nm from a previously reported study.28 Supporting Figure S1 shows the TGA curve of poly(MPC)-protected Fe3O4 nanoparticles. Poly(MPC) brushes decomposed at 200–450 °C, with a mass of 16.6%. The grafting density of poly(MPC)-protected on Fe3O4 was calculated from equation (1). The density of poly(MPC) was estimated as 0.16 chains/nm2; although this value is higher, it shows a good agreement with that reported previously.24 The density of poly(MPC) brushes was sufficient for reducing the nonspecific protein adsorption and cell adhesion. Figure 4 shows the effect of surface modification on the colloidal stability of Fe3O4 nanoparticles in diluted human serum. Bare Fe3O4 nanoparticles were prepared by hydrothermal synthesis. Just after preparing the suspension, high surface hydrophilicity and good dispersion in aqueous media were observed. However, bare nanoparticles completely precipitated after 3 h. The dispersion ability of Fe3O4 nanoparticles was well improved by the immobilization of poly(MPC) brushes. This tendency is in good agreement with that reported previously.25 Various water-soluble polymers have been used to improve the colloidal stability of nanoparticles. In particular, the stabilization of poly(MPC)-protected nanoparticles possibly contributes


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to their strong hydration layer via electrostatic interactions and the superiority of poly(MPC) in the stabilizing ability compared to neutral ethylene oxide.29,30 Figure 5A shows the average size increment (∆Size) of poly(MPC)-protected nanoparticles in contact with CRP at different concentrations. Blue and red closed circles represent the change in the nanoparticle size just after mixing with CRP and the change in the nanoparticle size after the redispersion of precipitated nanoparticles/CRP mixture using a neodymium magnet, respectively. The ∆Size of poly(MPC)-protected nanoparticles in contact with CRP increased with an increase in the concentration of CRP.

The driving force of the aggregation is the crosslinking of the nanoparticles with

CRP because CRP comprises five subunits. Very recently, Wu and co-workers have reported that the water molecules are released from phosphorylcholine moieties during CRP binding.31 The dehydration phenomenon may influence the colloidal stability of complexes of the nanoparticles with CRP and encourage to form the aggregation.

At

concentrations ranging from 0 to 100 nM, the size increased linearly. In particular, the limit of detection of CRP with poly(MPC)-protected Fe3O4 nanoparticles was improved by redispersion (red line).

The inter-particle binding of poly(MPC)-protected Fe3O4

nanoparticles through the CRP might be encouraged by the agglutination with a neodymium magnet. Then, the size increment of re-dispersed complexes of nanoparticles and CRP could be detected even at low concentration of CRP. A low limit of detection of 10 nM for CRP was nearly equal to the median concentration of CRP in healthy young adult volunteer blood donors.32 A good correlation between the ∆Size and CRP concentration was observed at CRP concentrations ranging from 0 to 600 nM (Supporting Figure S2). According to Figure 5B, the incremental manner is due to aggregation of poly(MPC)-protected Fe3O4 nanoparticles.


Previously, our group has

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reported the detection of CRP with MPC-block-copolymer-bearing gold nanoparticles.23 A detection limit of 40 nM is obtained for CRP using gold nanoparticles. In contrast, 10 nM of CRP was detected using poly(MPC)-protected Fe3O4 nanoparticles. The density of the block copolymers on gold nanoparticles was approximately one-fourth of that of poly(MPC) on the Fe3O4 nanoparticles prepared herein. High-density poly(MPC) exhibited preferable binding affinity to CRP. Furthermore, the black closed triangles shown in Figure 5 represent the ∆Size of poly(MPC)-protected nanoparticles in contact with CRP of various concentrations in the presence of a BSA concentration of 0.35 g/dL (≈53 µM). Even though the BSA concentration was approximately 1000 times greater than that of CRP, the nanoparticle size was not considerably different with and without BSA at each of the concentrations. This result indicated that poly(MPC)-protected Fe3O4 nanoparticles selectively bind to CRP. The decrease in the adsorption of proteins on MPC polymers has been well examined.33,34 Nonspecific protein adsorption induces an unfavorable host response and decreases the sensor capacity of diagnostic devices. Generally, proteins are adsorbed on a surface within a few minutes of the material coming in contact with body fluids such as blood, plasma, and tears. The adsorption of proteins on MPC polymers from human plasma determined by a radioimmunoassay and an immunogold-colloid labeling technique revealed that the adsorption of plasma proteins is effectively decreased regardless of the protein species.35 Figure

6

shows

the

repeated

aggregation

and

dissociation

of

poly(MPC)-protected Fe3O4 nanoparticles in contact with 30 nM of CRP/1 mM Ca2+ and 10 mM of EDTA, respectively. The binding of CRP to PC requires a conformational change in intact pentraxin, which is triggered by the binding of two free Ca2+ ions per subunit.36 When poly(MPC)-protected Fe3O4 nanoparticles were in contact with 30 nM


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of CRP in the presence of 1 mM Ca2+, an ~80-nm size increment of the nanoparticles was observed. In stark contrast, the nanoparticle size was restored to its original size after treatment with 10 mM of EDTA. This size change was reversible and reproducible. The poly(MPC)-protected Fe3O4 nanoparticles would be then applicable for the repeated detection of CRP. Figure 7 shows the specific collection of CRP with poly(MPC)-protected Fe3O4 nanoparticles. A solution comprising 12.5 nM of CRP and 20 nM of BSA was incubated with poly(MPC)-protected Fe3O4 nanoparticles for 2 h in the MES buffer. After the nanoparticles were precipitated using a neodymium magnet and further subjected to centrifugation, the concentration of each protein was determined by ELISA. The concentration of CRP in the supernatant became almost zero. In contrast, the BSA concentration did not change even after 2-h incubation. This result indicated that CRP can be collected by poly(MPC)-protected Fe3O4 nanoparticles without decreasing the concentration of coexisting BSA. In addition, the inhibition test for the selective binding of poly(MPC)-protected Fe3O4 nanoparticles with CRP in the presence of DOPC liposome37 or free poly(MPC) as the inhibitor was carried out (Figure 8). The concentration of CRP in the MES solution prepared for the test was 100 nM. When the concentrations of the phosphatidylcholine groups of DOPC liposomes and free poly(MPC) in the media were adjusted to be similar to the PC groups of poly(MPC)-protected

Fe3O4

nanoparticles,

the

elimination

of

CRP

with

poly(MPC)-protected Fe3O4 nanoparticles was not inhibited, indicating that the PC groups of poly(MPC) grafted on Fe3O4 nanoparticles preferentially bind to CRP compared to the phosphatidylcholine groups of DOPC liposomes and free poly(MPC). Kamon et al. have investigated the effect of thickness and grafting density of the MPC


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polymer brushes on self-assembled monolayer substrates for the selective binding of CRP.38 They have reported that controlling the poly(MPC) brush density rather than the layer thickness is the dominant factor to achieve high sensitivity for CRP. Therefore, poly(MPC)-protected Fe3O4 nanoparticles exhibit higher affinity to CRP compared to free poly(MPC). Goda et al. have investigated the effect of the chemical structure of phospholipids on the binding of CRP.39 On supported phospholipid monolayers comprising the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) monolayer, CRP did not bind to the surface in the absence of Ca2+. In contrast, the calcium-independent binding of CRP was observed on POPC/lysophosphatidylcholine composite monolayers. Furthermore, the affinity of phosphocholine to CRP is greater than that of glycerophosphorylcholine. To bind CRP with PC molecules, the chemical structure connecting the PC group is also important. Although additional experiments for understanding the difference in the binding mechanisms of CRP to phosphatidylcholine and MPC are required, poly(MPC) brush surfaces are confirmed to preferentially recognize CRP without being affected by impurities. Indeed, CRP was eliminated from a cell culture medium containing fetal bovine serum (EGM™-2 BulletKit™; Lonza, Switzerland) as shown in Supporting Figure S3. The CRP concentration dramatically decreased after treatment with poly(MPC)-protected Fe3O4 nanoparticles.

CONCLUSION In this study, the label-free detection and collection of CRP with

poly(MPC)-protected Fe3O4 nanoparticles was achieved. Poly(MPC)-protected Fe3O4 nanoparticles exhibited excellent colloidal stability in simulated body fluids because of


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the surface decoration of highly hydrated dense polymer brushes. The poly(MPC) brush structure was preferentially bound to CRP compared to phospholipids and free poly(MPC). Therefore, the size increment of the nanoparticles is well correlated with the CRP concentration, and it is not affected by coexisting BSA. Moreover, CRP was successfully eliminated from human plasma. The poly(MPC)-protected Fe3O4 nanoparticles can serve as useful, rapid, and simple tools for the detection of inflammation and control of CRP levels in plasma.

ASSOCIATED CONTENTS

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXXXX. Thermogravimetric analysis curves. Size increment of poly(MPC)–Fe3O4 after contact with various concentrations (0–600 nM) of CRP for 60 min. Concentration of CRP in the cell culture medium before and after treatment with poly(MPC)-protected Fe3O4 nanoparticles. (PDF)

AUTHOR INFORMATION

Corresponding author *(Y.I.) E-mail: [email protected]. Phone: +81-6-6368-0090. ORCID Yasuhiko Iwasaki: 0000-0003-1603-6174 Notes The authors declare no competing financial interest.


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ACKNOWLEDGEMENTS

This work was supported by the MEXT Private University Research Branding Project and AMED S-innovation Program for the development of biofunctional materials for the realization of innovative medicine.

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Figure Legends Figure 1

Preparation of poly(MPC)-protected Fe3O4 nanoparticles.

Figure 2

ATR-IR

spectra

BDCS-immobilized

of

bare Fe3O4

Fe3O4

nanoparticles

nanoparticles

(blue

(black

line),

line),

and

poly(MPC)-protected Fe3O4 nanoparticles (red line). Figure 3

Size distribution of bare Fe3O4 nanoparticles (black line) and poly(MPC)-protected Fe3O4 nanoparticles (red line).

Figure 4

Dispersion stability of bare Fe3O4 nanoparticles and poly(MPC)-protected Fe3O4 nanoparticles in 50% human serum.

Figure 5

(A) Change in the size of poly(MPC)-protected Fe3O4 nanoparticles in contact with CRP for 60 min at pH 5.5 and [Ca2+] = 1 mM (n = 3). Blue circle: only just mixed nanoparticles with CRP; red circle: resuspended agglutination formed using a neodymium magnet; black triangle: with 0.35 g/dL albumin under conditions similar to those shown in the red circle. (B) TEM images of poly(MPC)-protected Fe3O4 nanoparticles and those in contact with 100 nM of CRP for 60 min.

Figure 6

Repeated aggregation and dissociation of poly(MPC)-protected Fe3O4 nanoparticles in contact with 30 nM of CRP/1 mM of Ca2+ and 10 mM EDTA, respectively (n = 3).

Figure 7

Concentrations of albumin and CRP in the mixed protein solution before and after treatment with poly(MPC)-protected Fe3O4 nanoparticles (n = 3). Black: albumin; red: CRP. *p < 0.001 (t-test).

Figure 8

Inhibition test for the binding of CRP to poly(MPC)-protected Fe3O4 nanoparticles by the addition of DOPC liposomes or free poly(MPC) (n =


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3). The PC unit concentration of contaminants was equal to that of poly(MPC)-protected Fe3O4 nanoparticles. *p < 0.003 (t-test) vs before treatment with poly(MPC)-protected Fe3O4 nanoparticles. **p > 0.5 (t-test)

Figure S1

vs no contaminants.

Thermogravimetric analysis curves. Black line: bare Fe3O4 nanoparticles; red line: poly(MPC)-protected Fe3O4 nanoparticles.

Figure S2

Size increment of poly(MPC)–Fe3O4 after contact with various concentrations (0–600 nM) of CRP for 60 min (n = 3). Blue circle: only just mixed nanoparticles with CRP; red circle: resuspended agglutination formed using a neodymium magnet.

Figure S3

Concentration of CRP in the cell culture medium before and after treatment with poly(MPC)-protected Fe3O4 nanoparticles (n = 3). *p

Label-Free Specific Detection and Collection of C-Reactive Protein

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