Assembly of Plasmonic and Magnetic Nanoparticles with Fluorescent Silica Shell Layer for Tri-functional SERS-Magnetic-Fluorescence Probes and Its Bioapplications

In this study, we report on the fabrication of multilayered tri-functional magnetic-SERS-fluorescence nanoprobes (MF-SERS particles) containing clustered superparamagnetic Fe3O4 nanoparticles (NPs), silver NPs, and a fluorescent silica layer. The MF-SERS particles exhibited strong SERS signals from the silver NPs as well as both superparamagnetism and fluorescence. MF–SERS particles were uptaken by cells, allowing successful separation using an external magnetic field. SERS and fluorescence signals could be detected from the NP-containing cells, and CD44 antibody-conjugated MF-SERS particles selectively targeted MDA-MB-231 cells. Based on these properties, MF-SERS particles proved to be a useful nanoprobe for multiplex detection and separation of cancer cells.


Results and Discussion
Synthesis of MF-SERS particles. The MF-SERS particles were synthesized by introducing multiple functional layers, including clustered Fe 3 O 4 NPs, assembled Ag NPs, and a fluorescent dye-conjugated silica shell layer to provide SERS-magnetic-fluorescence tri-functionality, followed the conjugation of an antibody for biotargeting (Fig. 1a). The layers were composed of Fe 3 O 4 NP clusters that provided a strong response to an external magnetic field due to their superparamagnetism, and assembled Ag NPs for a strong SERS signal triggered by the formation of hot spots. Figure 1b shows the synthetic procedure for preparing the MF-SERS particles. Silica NPs were used as a backbone structure to immobilize the NPs, as they can be synthesized and modified easily. The silica NPs were synthesized by the Stöber method with a narrow size distribution (250 ± 25 nm), as shown in Figs 2a and S1a 39 . When the silica NPs were synthesized, their yield was approximately 31.3% (500 mg). Amine groups were then introduced onto the silica NPs using APTS. To immobilize the superparamagnetic Fe 3 O 4 NPs on the surface of the silica NPs, amine-functionalized silica NPs were coupled with caffeic acid to introduce catechol groups, which are known to have a strong affinity for Fe 3 O 4 NPs 40 . We used superparamagnetic NPs with an average diameter of 18 nm. They were well dispersed and displayed a uniform size. (Fig. S2a). To confirm the magnetic properties of the Fe 3 O 4 NPs, the field-dependent magnetization was measured at 300 K (Fig. S2b). The magnetization curve exhibited a saturated magnetization of 36 emu/g without coercivity, indicating that the Fe 3 O 4 NPs were superparamagnetic. To avoid aggregation of the Fe 3 O 4 NPs in the amphiphilic solvent when reacting with catechol-functionalized silica NPs, oleate-stabilized Fe 3 O 4 NPs underwent a ligand exchange process. Oleatestabilized Fe 3 O 4 NPs were treated with PVP at 100 °C, and then cooled to room temperature. The resulting Fe 3 O 4 NPs showed good dispersion in amphiphilic solvents, which confirms that the oleate ligands were replaced by PVP. The PVP-stabilized Fe 3 O 4 NPs were then mixed with the catechol-functionalized silica NPs to immobilize the Fe 3 O 4 NPs on the silica NPs. A silica layer with a thickness of 10 nm was then introduced onto the Fe 3 O 4 NP-embedded silica NPs to allow further surface modification (Figs 2b and S1b), and the resulting silica-coated Fe 3 O 4 NP-embedded silica NPs (M-SiO 2 NPs) were successfully synthesized similar to the previous results 34,41 . And, when they were synthesized, their yield was 12.8% (5.6 mg from 4 mg of caffeic acid modified SiO 2 with 12.5 mg of Fe 3 O 4 NPs.).
Next, the surface of the M-SiO 2 NPs was functionalized with thiol groups using MPTS to attract the Ag ions and aid the formation of Ag NPs 42 . Ag ions were reduced using octylamine, and enormous amounts of spherical Ag NPs (27 ± 3.2 nm) were assembled on the surface of the M-SiO 2 NPs (Figs 2c and S1c, S3). Thiol group containing aromatic compounds were used as RLCs. The surface of the Ag NPs-assembled M-SiO 2 NPs (Ag-M-SiO 2 NPs) was treated the RLCs, and then coated with silica to provide chemical stability and biocompatibility. To introduce the fluorescent shell, RITC was conjugated with APTS. Finally, approximately 0.8 mg MF-SERS particles were obtained 43 . The silicate monomer-conjugated RITC was then reacted with the silica-coated Ag-M-SiO 2 NPs. The thickness of the outer silica layer of the NPs was 17.19 ± 1.4 nm, as measured from a TEM image  Characterization of the MF-SERS particles. In order to provide MF-SERS particles with multiplex abilities, four RLCs (4-FBT, 4-CBT, 4-BBT, and 3,4-DCT) were used. SERS signals were obtained from the RLC-treated MF-SERS particles, and the unique SERS patterns of the respective molecules could be clearly distinguished by their narrow bands at 386 cm −1 (4-FBT), 488 cm −1 (4-BBT), 541 cm −1 (4-CBT), and 565 cm −1 (3,4-DCT) (Fig. 3a). Among the RLCs, even asymmetric aromatic compound (3,4-DCT), which usually gives low SERS signals, could also generate strong SERS signals due to the bumpy structure of Ag NPs layer of the MF-SERS particles. Thus, various aromatic compounds can be used as RLCs, expanding the multiplexing capability of MF-SERS particles.
The extinction spectra of NPs during every synthetic step for MF-SERS particles fabrication are shown in Fig. 3b. Compared to the absorption band of the M-SiO 2 NPs, the Ag-M-SiO 2 NPs showed an absorption band at ca. 400 nm and a broad band ranging from visible to NIR region. This was mainly due to the plasmonic property of the Ag NPs and their aggregate. This shows that the Ag NPs were well preserved after the silica shell coating step, as confirmed by TEM (Fig. 2d) and plasmonic properties of the Ag NPs were preserved during the NP's synthesis.
We also analyzed the photoluminescence spectrum of the MF-SERS particles with a 540 nm photo-excitation (Fig. 3c). An emission band at 580 nm was observed from MF-SERS particles, corresponding to the emission band of RITC, indicating that fluorescent RITC molecule was well introduced in the silica shell layer.
To confirm the reproducibility of the MF-SERS particles, three batches of particles were synthesized, and the absorbance at 430 nm and PL intensity at 540 nm were measured (Fig. S5a). Additionally, the MF-SERS particles were consistently well dispersed in several solvents, including ethanol, PBS (pH 7.4) and cell culture media (Fig. S5b). The results indicate that MF-SERS particles are dispersed in the silica layer and are suitable for cell studies.
To confirm the magnetic properties of the MF-SERS particles, the field-dependent magnetization was measured at 300 K (Fig. 3d). The magnetization curve exhibited a saturated magnetization of 2.1 emu/g without coercivity, indicating that the MF-SERS particles were superparamagnetic. In addition, MF-SERS particles were attracted to a magnet within 10 min, which is more advantageous for cell separation than single magnetic NPs (Fig. 3d, inset).

Cellular binding of MF-SERS particles.
Several studies have been reported on the interaction of NPs (~400 nm) with cells. NPs of approximately 400 nm size are known to bind to cells with about 50% being absorbed into the cell 44 . In addition, NPs with positive surface charges can have strong interactions with cells because the surface of the cells is negatively charged 45 . Thus, the surface of the MF-SERS particles was modified with APTS. The amine-functionalized MF-SERS particles (MF-SERS particles amine ) were then incubated with MDA-MB-231 cells on a glass slide at 37 °C for 2 h. The cells with and without MF-SERS particles amine were visualized by confocal microscopy to evaluate whether cellular binding or uptake had occurred (Fig. 4a). After proper washing, significant amount of orange fluorescence was observed from the MF-SERS particles amine around the nuclei of the MDA-MB-231 cells (blue). In addition, when various amounts of MF-SERS particles amine were treated to MDA-MB-231 cells, and the cells were analyzed by FACS, the intensities of the fluorescently-labeled cells were increased by increasing the amount of MF-SERS particles amine (Fig. S6). These results suggest that the MF-SERS particles amine (~400 nm) could bind to the cell surface.
SERS signals from MF-SERS particles bound cells were detected using point-by-point mapping using 660 nm laser excitation at a power of 11.8 mW with a step size of 1 µm and an exposure time of 1 s per point. The SERS map was then overlaid with the corresponding bright-field optical image, as shown in Fig. 4b. The SERS spectrum of the 4-FBT, that had been labeled as a RLC, could be obtained from regions (i), (ii), and (iii) in Fig. 4b. The SERS intensity in the SERS map was based on the height of the most intense peak of the 4-FBT spectrum, at 1075 cm -1 . As a result, SERS signals from MF-SERS particles could be collected even from cells.
Next, we attempted to separate the MF-SERS particles bound cells using an external magnetic field. The MF-SERS particles amine were mixed with MDA-MB-231 cells that were floating freely in the cell culture medium at 37 °C for 2 h. Then, a magnet was placed at the side of the cell mixture until the cells were pulled toward the magnet, as shown in Fig. 4c. The pulled down cells were collected and analyzed by fluorescence-activated cell sorting (FACS). As a result, cells with enhanced fluorescence emission were separated by FACS, as shown in Fig. 4c (population ii). Because the cells contain many MF-SERS particles, they had stronger fluorescence intensity than untreated MDA-MB-231 cells (Population i in Fig. 4c), resulting in a shift to the right in the FACS analysis. Furthermore, pure MF-SERS particles were analyzed by FACS, and the results were compared to those obtained with the MDA-MB-231 cells (Fig. S7). No particles were detected by FACS, showing that MF-SERS particles without interaction with cells during incubation period cannot be detected by FACS. Thus, these results indicate that MF-SERS particles bound cells were readily separated by external magnetic field. We also carried out a cell viability assay to evaluate the cytotoxicity of MF-SERS particles (Fig. S8). Dosages of MF-SERS particles at concentrations used in this study (from 0.1 to 10 µg/mL) showed a level of cell viability similar to the untreated group (0 µg/mL). These results indicate that there is no cytotoxicity of MF-SERS particles treated into the cell.

Specific binding of CD44 antibody-conjugated MF-SERS particles to MDA-MB-231 cells. The
antigen-specific binding of the CD44 antibody-conjugated MF-SERS particles (MF-SERS particles Ab ) to CD44-expressing cells was investigated. First, the surface of the MF-SERS particles was modified via EDC/ NHS coupling reaction in order to immobilize antibodies on the MF-SERS particles 46 . Briefly, the surface of the MF-SERS particles was functionalized with amine groups using APTS. Then, the amine groups on the surface of the MF-SERS particles were reacted with succinic anhydride to transform to carboxyl groups. The carboxyl groups were then activated by EDC/NHS for CD44 antibody coupling. After the CD44 antibody was conjugated to carboxyl groups, and the resulting MF-SERS particles Ab were incubated with CD44-expressing MDA-MB-231 cells at 4 °C for 2 h. A schematic illustration of the antigen-specific binding of the MF-SERS particles with the CD44-expressing cells is shown in Fig. 5a. We examined the CD44 expression in MDA-MB-231 or HepG2 cells by immunostaining by green fluorescence (Fig. S9). The green fluorescence for the CD44 antigen was not observed in the HepG2 cells, while strong green fluorescence clearly was observed in the MDA-MB-231 cells.
In the MDA-MB-231 cells, the orange fluorescence of the MF-SERS particles Ab was also clearly observed at the periphery of the cells with pseudo-blue fluorescent nuclei (Fig. 5b). Additional fluorescent cell images using confocal Z-stack acquisition were also obtained to demonstrate that the location of MF-SERS particles Ab appeared as orange or red fluorescence inside MDA-MB-231 cells ( Fig. S10 and Movie S1 in Supplementary information). As shown in Fig. S11 and movie clips (Movies S2 and S3 in Supplementary Information), orthogonal images from XZ, YZ, and XY projections with different Z-axis clearly demonstrate that MF-SERS particles Ab were internalized into the MDA-MB-231 cells. However, the orange fluorescence was rarely observed due to the absence of CD44 antibody conjugation or the CD44-negative HepG2 cells. These results prove that the MF-SERS particles Ab selectively recognized the CD44 antigen in the CD44-expressing MDA-MB-231 cells.
MF-SERS particles containing magnetic, fluorescence, and SERS properties were fabricated by immobilizing superparamagnetic Fe 3 O 4 NP clusters on silica NPs, assembling Ag NPs on them, and introducing a fluorescent silica layer. SERS signals were successfully obtained from aromatic RLCs (4-FBT, 4-CBT, 4-BBT, and 3,4-DCT) coated on the MF-SERS particles, and fluorescence signals were also obtained at the same time. The MF-SERS particles exhibited a strong response to an external magnetic field due to their superparamagnetic property. When cells were treated with the MF-SERS particles, the NP-bound cells could be separated from the others using external magnetic field and measured by FACS analysis. The characteristics of each modality of the MF-SERS particles, including fluorescence, SERS, and magnetic properties, were preserved after cellular uptake. Moreover, the MF-SERS particles could be modified with the CD44 antibody via an amide coupling reaction, and successfully targeted the CD44-positive cells. The tri-functional particles with SERS, magnetic, and fluorescent properties are expected to be useful nanoprobes for cell separation and multiplexed detection.

Methods
Chemical and materials. All reagents were used as received from the suppliers without further purification.  Then, the reaction mixture was cooled to 25 °C and poured slowly into 10 mL of diethyl ether. The mixture was centrifuged (4500 rpm, 5 min) then re-dispersed in EtOH. The silica NPs were synthesized by the Stöber method. TEOS (1.6 mL) and NH 4 OH (4 mL) were added to abs. EtOH (40 mL) and stirred for 20 h at 25 °C. The reaction mixture was washed several times with EtOH by centrifuging at 7000 rpm for 15 min. To introduce amine groups onto the surface of the silica NPs, silica NPs (40 mg in 20 mL EtOH) were incubated with APTS (100 μL) and NH 4 OH (100 μL) for 18 h at 25 °C. The reaction mixture was washed several times with EtOH, and then, re-dispersed in NMP.
The amine-functionalized silica NPs (20 mg in 5 mL DMF) were mixed with caffeic acid (7.2 mg) and one equivalents of HBTU, HOBt, and DIEA, and then reacted for 3 h at 25 °C. The reaction mixture was washed several times with DMF.
The catechol-functionalized silica NPs (1 mg in 5 mL DMF) and the PVP-stabilized Fe 3 O 4 NPs (0.4 mg in 5 mL EtOH) were mixed and sonicated for 1 h at 25 °C, and then, the reaction mixture was washed several times with EtOH.
For the silica coating, TEOS (50 μL) and NH 4 OH (100 μL) were added to the Fe 3 O 4 NP-embedded silica NPs (1 mg in 5 mL EtOH) and reacted for 18 h at 25 °C.
For introduction of thiol groups onto the surface of the M-SiO 2 NPs, the M-SiO 2 NPs (1 mg in 1 mL EtOH) were mixed with MPTS (50 µL) and NH 4 OH (10 µL) and reacted for 1 h at 50 °C. The reaction mixture was washed several times with EtOH.  Ag-M-SiO 2 NPs (in 2 mL IPA), along with water (400 µL), TEOS (2 µL), and NH 4 OH (10 µL). The mixture was incubated for 18 h at 25 °C, and the resulting fluorescence shell-coated Ag-M-SiO 2 particles (MF-SERS particles) were washed several times with EtOH.
For introduction of amine groups, the MF-SERS particles (0.5 mg in 1 mL EtOH) were incubated with APTS (50 µL) and NH 4 OH (10 µL) and reacted for 1 h at 25 °C. The reaction mixture was washed several times with EtOH, and the resulting amine-functionalized MF-SERS particles (MF-SERS particles amine ) were re-dispersed in PBS buffer solution (10 mM, pH 7.4).
For introduction of antibodies onto the MF-SERS particles, MF-SERS particles amine were re-dispersed in NMP (0.5 mL) and reacted with succinic anhydride (1.75 mg) and DIEA (3. Characterization of the MF-SERS particles. Transmission electron microscope (TEM) images of NPs were obtained using a Carl Zeiss LIBRA 120 (Oberkochen, Germany), and a JEOL JEM-3000F (Tokyo, Japan) was used for energy-dispersive X-ray spectroscopy (EDX) mapping imaging analysis. SERS measurements were performed using a micro-Raman system (LabRam 300, JY-Horiba). Extinction properties of NP samples were analyzed using a UV/vis spectrophotometer (Mecasys OPTIZEN POP, Daejeon, Korea). Photoluminescence intensities were obtained using a fluorescence spectrophotometer (Model Cary Eclipse, Agilent Technologies, Santa Clara, CA, USA). Field-dependent magnetization of dried MF-SERS particles was measured using a PPMS-14 (Quantum Design, USA). Fluorescence microscopic images were obtained using a confocal laser scanning microscope (Olympus FV-1000 spectral, Tokyo, Japan).

Cell culture and internalization of MF-SERS particles. MDA-MB-231 cells (breast cancer epithelial
cell line, purchased from American Type Culture Collection, ATCC HTB-26) and HepG2 cells (liver epithelial cell line, purchased from ATCC, ATCC HB-8065) were cultured in Dulbecco's modified Eagle's medium with high glucose (HyClone Laboratories, Logan, UT, USA) supplemented with 10% fetal bovine serum (HyClone Laboratories) and 100 U/mL of penicillin (Welgene, Daegu, Korea) at 37 °C in humidified air containing 5% CO 2 . To determine the cellular binding of MF-SERS particles amine to the cells, MDA-MB-231 cells were seeded onto a 24-well plate with cover glass (Paul Marienfeld GmbH, Lauda-Königshofen, Germany) at a density of 2.0 × 10 5 cells and incubated at 37 °C. After 16 h of incubation, the cover glass was blocked with 3% bovine serum albumin (BSA) in PBS at room temperature for 30 min. After washing with PBS twice, 500 μL of media containing 0.25 mg/mL of MF-SERS particles amine was added to the cells and incubated again at 37 °C for 2 h. The cells were washed twice with PBS and fixed with 4% paraformaldehyde (w/v) at room temperature for 1 h. The cells were washed again with PBS, and the nuclei were stained using TOPRO-3 (1:1000; T3605, Invitrogen, Carlsbad, CA, USA) diluted in PBS at room temperature for 30 min. The cells were then mounted on microscope slides (Paul Marienfeld GmbH) with ProLong Gold antifade reagent (Invitrogen) and observed using a confocal laser scanning microscope (Olympus FV-1000 spectral, Tokyo, Japan). The orange fluorescence from the MF-SERS particles amine and the pseudo-blue fluorescence from TOPRO-3 stained nuclei were monitored and merged with the bright field cell images. In addition, SERS signal from the MF-SERS particles amine -treated cells mounted on microscope slides were obtained by point-by-point mapping using a 660 nm laser line at the power of 11.8 mW with a 1-µm step size for 1 s. Magnetic isolation and flow cytometry analysis. Fresh cell culture medium (2.5 mL) was prepared in a 5 mL tube (Eppendorf, Hauppauge, NY, USA), containing MDA-MB-231 cells (2.0 × 10 5 ) with or without MF-SERS particles amine (20 μL, 0.25 mg/mL). After incubation at 37 °C for 2 h, a strong magnet (4000 gauss) was placed on one side of the tube, followed by a further 2 h incubation at room temperature. The cells submerged at the bottom of the tube were carefully removed, and only the cells attracted to the wall by the magnet were collected and quantified by fluorescence-activated cell sorting (FACS) analysis using a FACSCalibur Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Antibody-specific binding of MF-SERS particles on MDA-MB-231 cells.
The specific binding of the MF-SERS particles Ab to antigen-expressing cells was monitored by observing orange fluorescence of the MF-SERS particles Ab on the cells using a confocal microscope. The CD44-positive (+) cell line (MDA-MB-231 cells) and the CD44-negative (−) cell line (HepG2 cells) were grown in the wells of a 12 well plate with cover glass at a density of 1.0 × 10 6 cells. The cells were fixed with 4% paraformaldehyde at room temperature for 1 h and blocked with 3% BSA. After washing three times with PBS, 1 mL of PBS containing MF-SERS particles Ab (0.25 mg/mL) or MF-SERS particles (0.25 mg/mL) were added to the cells and incubated at 4 °C for 2 h. The primary CD44 antibody (1:1000 dilution) and fluorescence-conjugated secondary antibodies (1:1000; Alexa Fluor 488) were also treated to MDA-MB-231 cells or HepG2 cells at 4 °C for 2 h as a control (Fig. S9). The cells were washed three times with PBS and visualized using a confocal microscope after nuclei staining as mentioned above.