2024 Volume 72 Issue 3 Pages 340-344
In clinical diagnosis, magnetic polystyrene nanoparticles (MPS NPs) are commonly applied to, e.g., the chemiluminescent immunoassay (CLEIA). However, the conventional preparation method of MPS NPs requires a long duration of heating to form polymer particles, which is inefficient. In this study, we prepared MPS NPs by emulsion solvent-evaporation without heating. We evaluated the effect of the solvent in the water and organic phases on the magnetic particle content. MPS NPs prepared by 4% (v/v) MeOH aqueous solution and adding stearic acid (SA) (4MeSA–MPS NPs) exhibited the highest magnetic particle content. Furthermore, CLEIA analysis indicates that the C-reactive protein detection limit is 80 pg/mL. Thus, 4MeSA–MPS NPs are promising for clinical diagnoses.
Magnetic materials such as iron and gadolinium have been applied to the biomedical,1,2) automotive,3,4) and storage media5,6) industries. For instance, Shen et al. developed a novel Fe–Si alloy that exhibited improved rotary torque, and is promising for motor applications.7) In the biomedical field, the application of magnetic materials has expanded to clinical diagnosis,8) contrast media,9) and hyperthermia.10) In the context of biocompatibility and cost, iron oxide is common. For example, Qian et al. reported that silk fibroin hydrogel containing iron oxide nanocubes had high anticancer activity against 4T1 cells under an alternating magnetic field.11) Garcia et al. developed iron oxide nanoparticle (NP)-loaded dental adhesives and showed high dentin’s bonding efficacy without cytotoxicity.12)
Regarding practicality, iron oxide NPs have been used as antibody carriers because of ease of separation with a magnet, resulting in shortened process times compared with centrifugation method for diagnosis.13) To prevent the release of iron ions from interfering with diagnoses, iron oxide NPs can be encapsulated in a polystyrene (PS) matrix, termed magnetic polystyrene (MPS) NPs. MPS NPs have been prepared by mini-emulsion14) and soap-free15) polymerization. However, these conventional preparation methods require a long duration of heating to form the polymer particles, which is inefficient.
Emulsion solvent-evaporation is a preparation method for polymer particles that does not require heating.16,17) Ling et al. developed uniform dispersed iron oxide encapsulated poly (lactic-co-glycolic acid) (PLGA) NPs by emulsion solvent-evaporation and applied them to magnetic resonance imaging as well as cancer therapy.18) However, in emulsion solvent-evaporation, most studied polymers are biodegradable; such as PLGA,18) polycaprolactone,19) and polylactic acid.20) Thus, uniform MPS NPs prepared by emulsion solvent-evaporation have not been reported in the literature.
In this study, we developed MPS NPs by emulsion solvent-evaporation and evaluated their fundamental physicochemical properties. Moreover, we applied the resulting MPS NPs to the chemiluminescent immunoassay (CLEIA) as one of the commonly utilized clinical diagnosis using MPS NPs and evaluated their applicability to clinical diagnoses (Fig. 1).
PS (Mw 280000) was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Iron oxide NPs (diameter: 10 nm) were purchased from Ferrotec Material Technologies (Tokyo, Japan). Chloroform (CHCl3), stearic acid (SA), and methanol (MeOH) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). C-Reactive protein (CRP)–monoclonal antibody (mAb) and CRP–immunoglobulin G (IgG) were purchased from Oriental Yeast (Tokyo, Japan). Horseradish peroxidase (HRP)-conjugated AffiniPure goat anti-rabbit (aRb) IgG was purchased from Jackson ImmunoResearch (West Grove, PA, U.S.A.).
Preparation of MPS NPs by Emulsion Solvent-EvaporationMPS NPs were synthesized by emulsion solvent-evaporation in accordance with a previous report21) with major modifications. In brief, a CHCl3 solution (450 µL) consisting of iron oxide NPs (4.23 mg), PS (1.02 mg), and SA (45 µg) was added to 4 mL of H2O or 4% (v/v) aqueous MeOH at room temperature. The mixture was treated with an ultrasonic homogenizer under an ice bath for several minutes followed by incubating at room temperature overnight to evaporate the CHCl3. Finally, the resulting MPS NPs (3 mL) were washed with water 3× under a magnet and re-dispersed in water (3 mL). The MPS NPs were stored at room temperature.
Characterization of MPS NPsThe absorption spectrum was measured with a microplate reader (Infinite M200 PRO; Tecan Group, Männedorf, Switzerland). The size distribution and zeta potential of the particles were measured by a zetasizer (ELSZ-2000; Otsuka Electronics, Osaka, Japan). The shape and size of the particles were observed by field-emission electron probe micro analysis (FE EPMA), by scanning electron microscopy (SEM; JXA-8530F; JEOL, Tokyo, Japan). The magnetic particle content in the MPS NPs was measured by thermogravimetry–differential thermal analysis (TG8120; Rigaku, Tokyo, Japan).
Evaluation of Magnetic Properties of MPS NPsA vial of aqueous MPS NPs (3 mL) was fixed onto a magnet (10 × 10 × 10 mm, 0.42 T). The supernatant (100 µL) was collected every 30 s for 300 s. Each supernatant was transferred to a 96-well plate and the absorption at 400 nm was measured with a microplate reader.
CLEIA for CRP Detection with MPS NPsAqueous MPS NPs (200 µL) were washed with 10 mM bicine buffer and re-suspended in 11.6 mg/mL rCRP mAb (2 µL). The resulting mixture was shaken at room temperature for 1 h. rCRP mAb-adsorbed MPS NPs were prepared by washing with 1% bovine serum albumin (BSA) and 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (200 µL) followed by 1% BSA (200 µL). Next, each well of the 96-well plate was blocked by incubating with 1% BSA/MOPS (400 µL) at room temperature for 30 min and removed. rCRP–MPS NPs (25 µL) and 0–104 pg/mL rhCRP/1% BSA–4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (50 µL) were added to each well, and the mixture was shaken at room temperature for 3 min. The mixture was washed with pcAb/1%B SA (95 µL), 1% BSA (150 µL), and 0.1 µg/mL aRb–HRP/1% BSA (95 µL). Finally, each well was added to Wako ELISA-Star Mix (100 µL) and the luminescence intensity was measured.
Statistical AnalysisStatistical analyses were carried out by Student’s t-tests. The data are reported as mean ± standard deviation.
The advantage of emulsion solvent-evaporation is that a long duration of heating is not required to form the polymer particles, such as in the mini-emulsion14) and soap-free15) polymerization. In this study, we added MeOH to the water phase and expected to increase the content of magnetic particles in the MPS NPs because of the globule transition.22) First, we examined the relationship between the MeOH concentration in the water phase and MPS NP formation. We efficiently formed MPS NPs under 4% (v/v) aqueous MeOH (Supplementary Fig. S1). At >4% (v/v) MeOH, the surface tension of the water and organic phase might become excessive, which could hinder formation of emulsions. At <4% (v/v) MeOH, emulsions did not form after treating with sonication for 60 s. Next, we examined the relationship between that sonication time and MPS NP formation. The minimum sonication time for forming stable emulsions, in H2O and 4% aqueous MeOH, was 300 and 60 s, respectively (Supplementary Fig. S2). Thus, the optimized MeOH concentration and sonication time were 4% and 300 s, respectively. In the preparation of polymer particles, SA is mainly used as the dispersant. The alkyl moiety of SA is embedded into polymer matrix by hydrophobic interaction in aqueous solution. As a result, the negatively charged carboxy group is existed on the surface of polymer particles and improved dispersion stability of polymer particles. Meanwhile, the physicochemical property of polymer particles is known to be affected to various factors such as dispersant,23) polymer material,24) and solvent.25)
We prepared four types of MPS NPs combining the water and organic phases (Table 1).
| Abbreviation | Water Phase | Organic phase |
|---|---|---|
| H–MPS NPs | H2O | CHCl3 |
| HSA–MPS NPs | H2O | CHCl3 + SA |
| 4Me–MPS NPs | 4%MeOH aq. | CHCl3 |
| 4MeSA–MPS NPs | 4%MeOH aq. | CHCl3 + SA |
All of the MPS NPs were brown cloudy solutions (Fig. 2a). To examine the optical properties of each MPS NPs, the absorption spectra were measured. The absorbance of 4MeSA–MPS NPs and 4Me–MPS NPs in the visible light region was higher than that of H–MPS NPs and HSA–MPS NPs (Supplementary Fig. S3). Specifically, the absorbance of 4MeSA–MPS NPs at 400 nm was 1.9× higher than that of H–MPS NPs (Fig. 2b). To evaluate the shape and diameter of the MPS NPs, SEM was performed by FE EPMA. The obtained MPS NPs were nearly monodisperse in terms of spherical shape and diameter (Fig. 2c). To confirm the presence of Fe in the MPS NPs, elemental analysis was conducted. All the obtained MPS NPs contained Fe (Fig. 2d). Thus, we prepared MPS NPs by emulsion solvent-evaporation.
(a) Photographs. (b) Relative absorbance at 400 nm. (c) SEM images. (d) Elemental analysis.
To characterize the physicochemical properties of H–MPS NPs and 4MeSA–MPS NPs, the mean diameter and zeta potential were measured. Dynamic light scattering (DLS) indicates that the mean diameter of H–MPS NPs and 4MeSA–MPS NPs was 147.7 ± 55.2 and 117.9 ± 13.4 nm, respectively (Table 2). In general, the detection sensitivity is enhanced with decreasing the size of particles due to the increased surface area per particle volume and the increased antibody-analyte complex on the surface on particles. The zeta potential of H–MPS NPs and 4MeSA–MPS NPs was −8.6 ± 1.1 and −9.0 ± 0.8 mV, respectively. In terms of the mean diameter and zeta potential, there were no substantial differences between the H–MPS NPs and 4MeSA–MPS NPs (Supplementary Fig. S4). However, the magnetic particle content of 4MeSA–MPS NPs was 85.3% (w/w) and clearly increased compared with that of H–MPS NPs. The recovery rates of H–MPS NPs and 4MeSA–MPS NPs after a contact time of 150 s were 77 and 67%, respectively. The high recovery rate of H–MPS NPs may be due to the relatively large particle diameter compared with 4MeSA–MPS NPs in aqueous solution. This result may indicate that the larger magnetic particle size was increased the magnetic collection speed. To compare the physicochemical property of MPS NPs prepared by the conventional method, we prepared the MPS NPs by mini-emulsion polymerization method (Mini-MPS NPs). The resulting Mini-MPS NPs were spherical shape and successfully prepared (Supplementary Fig. S5). The magnetic particle content of Mini-MPS NPs was 80%(w/w) and lower than that of 4MeSA–MPS NPs, indicating that the magnetic collection ability and availability for CLEIA may be equivalent or less than that of 4MeSA–MPS NPs.
| Sample | Mean diameter (nm) | Zeta potential (mV) | Magnetic particle content (w/w%) | Recovery rate (%) |
|---|---|---|---|---|
| H–MPS NPs | 147.7 ± 55.2 | −8.6 ± 1.1 | 58.6 | 77 |
| 4MeSA–MPS NPs | 117.9 ± 13.4 | −9.0 ± 0.8 | 85.3 | 67 |
To evaluate the basis of the improved magnetic particle content, we focused on the PS structure in the emulsion. The PS structure changes from a coiled to globule structure (globule transition), by changing from good to poor solvent.26) In our emulsion solvent-evaporation system, MeOH (a poor solvent to PS) was in both the water and organic phases. Thus, the PS structure might be the cause of the globule transition in the emulsion, indicating that the PS network was more compact compared with that in the emulsion without MeOH (Fig. 3). In so doing, one can encapsulate magnetic particles in the PS matrix. Regarding the emulsion without MeOH, magnetic particles escaped from the loose PS matrix, leading to MPS NPs with low magnetic particle content. Because of the aforementioned PS structural change, 4MeSA–MPS NPs exhibited a higher magnetic particle content compared with H–MPS NPs.
To evaluate the potential of 4MeSA–MPS NPs for clinical diagnoses, we first examined the magnetic properties. We collected an increasing concentration of 4MeSA–MPS NPs with a magnet over increasing contact time (Fig. 4a). However, we observed neither aggregation nor precipitation of 4MeSA–MPS NPs without a magnet, indicating that the 4MeSA–MPS NPs were uniformly dispersed. To quantify the magnetic collection ability of the 4MeSA–MPS NPs, we measured the time-dependent absorbance of the supernatant. We collected 67 and 82% of 4MeSA–MPS NPs to the magnet after a contact time of 150 and 300 s, respectively (Fig. 4b). Thus, the 4MeSA–MPS NPs exhibited a fast magnetic response and have potential for clinical diagnoses.
(a) Photograph with or without a magnet. (b) Quantification of time-dependent magnetic collection (black: without magnet, red: with magnet).
Finally, we applied 4MeSA–MPS NPs to CLEIA and chose CRP as a model clinical infection marker. CL intensity was calculated by the fluorescence from the HRP conjugated to antibody-rCRP complex on magnetic particles. In general, as rCRP concentration was increased, CL intensity was proportionally increased.27) Therefore, we evaluated the detection limit using this system. The CRP detection limit is 80 pg/mL by using the 4MeSA–MPS NPs (Figs. 5a, b). The CRP concentration in human plasma under acute bacterial infection and rheumatoid arthritis increase from a value of 0.8 µg/mL to values of 31 and 73 µg/mL,28) respectively. Therefore, 4MeSA–MPS NPs might be useful in practical clinical diagnoses.
(a) Detection sensitivity. (b) Photograph of each measured well over various CRP concentrations.
We developed 4MeSA–MPS NPs by emulsion solvent-evaporation for clinical diagnoses. We optimized the magnetic particle content by adding MeOH in the emulsion solvent-evaporation system. The 4MeSA–MPS NPs exhibited a fast magnetic response and the CRP detection limit is 80 pg/mL. Thus, 4MeSA–MPS NPs are promising for CLEIA and other clinical diagnostic applications.
We acknowledge Mr. K. Kudo, Ms. N. Hanazawa, Mr. M. Sato, Mr. A. Awaji, Mr. S. Watanabe, and Dr. H. Shiigi of the Medical Material Team at the Tokuyama Corporation for their technical support.
The authors declare no conflict of interest.
This article contains supplementary materials.
