Silica-Coated Magnetic Iron Oxide Nanoparticles Grafted onto Graphene Oxide for Protein Isolation

In this study, silica-coated magnetic iron oxide nanoparticles (MNPs@SiO2) were covalently conjugated onto graphene oxide (GO/MNP@SiO2) for protein isolation. First, MNPs were precisely coated with a silica layer on the surface by using the reverse microemulsion method, followed by incubation with 3-glycidyloxypropyltrimethoxysilane (GPTS) to produce the GPTS-functionalized MNPs@SiO2 (GPTS-coated MNPs@SiO2) that display epoxy groups on the surface. The silica shell on the MNPs was optimized at 300 µL of Igepal®CO-520, 5 mg of MNP, 100 µL of TEOS, 100 µL of NH4OH and 3% of 3-glycidyloxypropyltrimethoxysilane (GPTS). Simultaneously, polyethyleneimine (PEI) was covalently conjugated to GO to enhance the stability of GO in aqueous solutions and create the reaction sites with epoxy groups on the surface of GPTS-coated MNP@SiO2. The ratio of PEI grafted GO and GPTS-coated MNP@SiO2 (GO/MNP ratio) was investigated to produce GO/MNPs@SiO2 with highly saturated magnetization without aggregation. As a result, the GO/MNP ratio of 5 was the best condition to produce the GO/MNP@SiO2 with 9.53 emu/g of saturation superparamagnetization at a magnetic field of 2.0 (T). Finally, the GO/MNPs@SiO2 were used to separate bovine serum albumin (BSA) to investigate its protein isolation ability. The quantity of BSA adsorbed onto 1 mg of GO/MNP@SiO2 increased sharply over time to reach 628 ± 9.3 µg/mg after 15 min, which was 3.5-fold-higher than that of GPTS-coated MNP@SiO2. This result suggests that the GO/MNP@SiO2 nanostructure can be used for protein isolation.


Characterization
Transmission electron microscope (TEM) images of the sample were taken using a Libra 120 field-emission transmission electron microscope (Carl Zeiss, German) with a maximum accelerated voltage of 120 kV. Optical properties of our materials were observed by an Optizen POP UV/Vis Spectrometer (Mecasys, Seoul, Korea). Magnetic measurement was carried out using a PPMS-9 vibrating sample magnetometer (Quantum Design, San Diego, CA, USA) at room temperature. Attenuated total reflection-Fourier transform infrared spectra of our materials was recorded on a Nicolet TM iS TM 50 Fourier transform infrared spectroscopy (FTIR) Spectrometer (Thermo Fisher, Waltham, MA, USA).

Effect of Igepal ® CO-520 Volume on Silica-Coated Magnetic Fe 3 O 4 Nanoparticles
MNPs were coated with silica by the reverse microemulsion method. Various volumes of Igepal ® CO-520 (0.1 to 5 mL) were added into 11 mL cyclohexane and sonicated for 10 min. Subsequently, 5 mg of MNPs in chloroform was added to the solution and mixed for several minutes to disperse the MNPs into the Igepal ® CO-520 solution. Next, NH 4 OH (100 µL) and TEOS (100 µL) were added drop-wise, and the mixture was incubated for 16 h at 25°C. Next, the mixture was precipitated by adding 20 mL EtOH and centrifuged at 8500 rpm for 30 min. The precipitate was washed five times with EtOH. Finally, the MNPs@SiO 2 were re-dispersed into EtOH to obtain a 1 mg/mL MNP@SiO 2 solution.

Effect of Quantity of MNPs on Silica-Coated Magnetic Fe 3 O 4 Nanoparticles
A volume of 0.3 mL of Igepal ® CO-520 was added into 11 mL cyclohexane and sonicated for 10 min. Subsequently, various quantities of MNPs in chloroform (1, 3, 5, 7 and 10 mg) were added to the solution and mixed for several minutes to disperse the MNPs into the Igepal ® CO-520 solution. Next, NH 4 OH (100 µL) and TEOS (100 µL) were added drop-wise, and the mixture was incubated for 16 h at 25°C. Next, the mixture was precipitated by adding 20 mL EtOH and centrifuged at 8500 rpm for 30 min. The precipitate was washed five times with EtOH. Finally, the MNPs@SiO 2 were re-dispersed into EtOH to obtain a 1 mg/mL MNP@SiO 2 solution.

Effect of TEOS Volume on Silica-Coated Magnetic Fe 3 O 4 Nanoparticles
A volume of 0.3 mL of Igepal ® CO-520 was added into 11 mL cyclohexane and sonicated for 10 min. Subsequently, 5 mg of MNPs in chloroform was added to the solution and mixed for several min to Nanomaterials 2020, 10, 117 4 of 14 disperse the MNPs into the Igepal ® CO-520 solution. Next, NH 4 OH (100 µL) and various volumes of TEOS (10, 50, 100, 150 and 200 µL) were added drop-wise, and the mixture was incubated for 16 h at 25 • C. Next, the mixture was precipitated by adding 20 mL EtOH and centrifuged at 8500 rpm for 30 min. The precipitate was washed five times with EtOH. Finally, the MNPs@SiO 2 were re-dispersed into EtOH to obtain a 1 mg/mL MNP@SiO 2 solution.

Preparation of Graphene Oxide Grafted with Polyethyleneimine (PEI grafted GO)
GO (2.5 mg in 0.5 mL H 2 O) was added to a PEI solution (400 µg PEI in 9.5 mL H 2 O) to obtain a final concentration of 40 µg/mL of PEI. The mixture was sonicated for 30 min and incubated at 60 • C for 12 h. Then, the mixture was centrifuged at 17,000 rpm for 1 h, and the precipitate was washed five times with PBS solution to remove the unbound PEI. PEI-adsorbed to GO was re-dispersed in a 0.5 mL PBS.
EDC (20 mg) and NHS (14 mg) in 0.5 mL PBS were added into the PEI-adsorbed GO solution to activate the carboxyl groups for 1 h. PEI (20 mg) was added to the mixture. The mixture was incubated for 2 h at room temperature to conjugate PEI onto the GO surface. An ethanolamine solution (100 µL, 1 M) was added to the mixture and incubated for 1 h to block the unreacted carboxyl groups. PEI grafted GO was collected by centrifugation at 17,000 rpm for 30 min and re-dispersed in 5 mL PBS.

Preparation of Silica-Coated Magnetic Nanoparticles Grafted onto Graphene Oxide (GO/MNP@SiO2)
GPTS-coated MNP@SiO 2 solution (250 µg) was added to the PEI-grafted GO solution (250 to 2500 µg) and vigorously mixed for 2 h at 25 • C. An ethanolamine solution (100 µL, 1.0 M) was added to the mixture and incubated for 1 h to block the unreacted epoxy groups. GO/MNP@SiO 2 was collected by centrifugation 3 times at 8500 rpm for 15 min, followed by applying a magnetic force for 5 min and washing 5 times with PBS.
2.6. BSA Isolation by GO/MNP@SiO 2 BSA was adsorbed into GO/MNP@SiO 2 by incubation. Briefly, 1 mg of GO/MNP@SiO 2 in PBS was incubated with 1% BSA in PBS buffer (100 µL) for 2 h. BSA adsorbed GO/MNP@SiO 2 was collected by applying a magnetic separator. The BSA adsorbed GO/MNP@SiO 2 was washed several times with PBS, and the supernatant was collected and diluted to 50 mL. The concentration of BSA in supernatant was determined by UV-Vis spectroscopy at 280 nm. Similarly, MNP@SiO 2 -GPTS was used as a control sample for BSA isolation.

Results and Discussion
Superparamagnetic MNPs (~20 nm) were coated with a silica layer using the reverse microemulsion method. The surface of MNP@SiO 2 was modified with epoxy groups by incubating them with GPTS (MNPs@ SiO 2 -GPTS). Simultaneously, PEI was first adsorbed on the GO surface for 24 h at 60 • C, and stabilized the GO surface so that GO can be well dispersed in an aqueous solution (PEI adsorbed GO). Subsequently, the COOH groups of the GO surface were coupled with the NH 2 groups of PEI using EDC/NHS (PEI grafted GO). Finally, GPTS-coated MNPs@SiO 2 were conjugated to the surface of PEI grafted GO by coupling the PEI NH 2 groups with the epoxy groups on the surface of GPTS-coated MNP@SiO 2 , producing GO/MNP@SiO 2 nanocomposites ( Figure 1).

Optimized Preparation of Silica-Coated Magnetic Nanoparticles (MNPs@SiO2)
The coating of the silica layer on the MNP surface via the reverse microemulsion method was optimized by varying experimental parameters such as Igepal CO-520 and TEOS volumes, as well as the number density of MNPs.

Optimized Preparation of Silica-Coated Magnetic Nanoparticles (MNPs@SiO 2 )
The coating of the silica layer on the MNP surface via the reverse microemulsion method was optimized by varying experimental parameters such as Igepal CO-520 and TEOS volumes, as well as the number density of MNPs.

Effect of Quantity of MNPs on Silica-Coated Magnetic Fe3O4 Nanoparticles
To obtain superparamagnetic MNPs@SiO2 without the presence of bare SiO2 NPs, the ratio of MNP and micelle number density is a critical parameter. Varying the MNP quantity (1 to 10 mg) revealed that bare SiO2 NPs were produced with 1 mg of MNPs ( Figure 3). The diameter of MNPs@SiO2 when 1 mg of MNP was used equaled 46.3 ± 1.1 nm, which is larger than the diameter of 37.7 ± 1.4 nm when 5 mg of MNP was used. This result indicates that the silica layer of MNPs is thicker and TEOS was excess in solution at 1 mg of MNP than at 5 mg of MNP. Along with the disappearance of the bare SiO2 NPs, the diameter of MNPs@SiO2 decreased with increasing quantity of MNP ( Figure 3c). However, at high quantity of MNP at 7 mg or 10 mg, the TEOS in solution was not enough to produce the silica layer on the surface of all MNPs. MNP@SiO2 was became irregular shape ( Figure 3a). Therefore, 5 mg of MNPs was chosen for further study.

Effect of Quantity of MNPs on Silica-Coated Magnetic Fe 3 O 4 Nanoparticles
To obtain superparamagnetic MNPs@SiO 2 without the presence of bare SiO 2 NPs, the ratio of MNP and micelle number density is a critical parameter. Varying the MNP quantity (1 to 10 mg) revealed that bare SiO 2 NPs were produced with 1 mg of MNPs ( Figure 3). The diameter of MNPs@SiO 2 when 1 mg of MNP was used equaled 46.3 ± 1.1 nm, which is larger than the diameter of 37.7 ± 1.4 nm when 5 mg of MNP was used. This result indicates that the silica layer of MNPs is thicker and TEOS was excess in solution at 1 mg of MNP than at 5 mg of MNP. Along with the disappearance of the bare SiO 2 NPs, the diameter of MNPs@SiO 2 decreased with increasing quantity of MNP ( Figure 3c). However, at high quantity of MNP at 7 mg or 10 mg, the TEOS in solution was not enough to produce the silica layer on the surface of all MNPs. MNP@SiO 2 was became irregular shape ( Figure 3a). Therefore, 5 mg of MNPs was chosen for further study.

Effect of TEOS Volume on Silica-Coated Magnetic Fe3O4 Nanoparticles
The volume of TEOS precursor is an important factor that affected on the thickness of silica shell of superparamagnetic MNP and the formation of bare SiO2 NP formation in the solution [46,48]. When TEOS is then added to the mixture, it converts to hydrolyzed TEOS adsorbed onto the superparamagnetic MNP surface, which results in transfer of the superparamagnetic MNPs into the water phase [46]. Finally, the hydrolyzed TEOS on the superparamagnetic MNP surface undergoes a condensation reaction to form the silica shell [46]. As the TEOS content increased from 10 to 100 µL, the diameter of MNPs@SiO2 and thickness of silica shell on the surface of MNP increased. It reached a plateau at 100 µL of TEOS in Figure 4. The silica layer was negligible at 10 µL, while the silica layer was not uniform at 50 µL of TEOS. The MNP@SiO2 with uniform thickness of silica layer on the surface was obtained at 100 µL of TEOS. The MNP@SiO2 diameter increased to 24.2 ± 2.0, 31.1 ± 2.3, and 37.7 ± 1.4 nm with 10, 50, and 100 µL of TEOS, respectively (Figure 4b). Increasing the volume of TEOS further caused the formation of bare SiO2 NPs, while the diameter of MNP@SiO2 was insignificantly different (Figure 4b,c). So, 100 µL of TEOS was chosen for further study.

Effect of GPTS Concentration on Silica-Coated Magnetic Fe3O4 Nanoparticles
To conjugate the MNPs@SiO2 onto the GO-grafted PEI, superparamagnetic MNPs@SiO2 must be modified with GPTS to create GPTS-coated MNPs@SiO2 that displayed epoxy groups on the surface, enabling conjugation with the NH2 groups of PEIs. The superparamagnetic MNPs@SiO2 were incubated in a toluene solution containing 1 to 3% GPTS for 1 h. The diameter remained unchanged and no bare SiO2 was obtained after GPTS treatment ( Figure S2). So, 3% GPTS was chosen for further study.

Effect of TEOS Volume on Silica-Coated Magnetic Fe 3 O 4 Nanoparticles
The volume of TEOS precursor is an important factor that affected on the thickness of silica shell of superparamagnetic MNP and the formation of bare SiO 2 NP formation in the solution [46,48]. When TEOS is then added to the mixture, it converts to hydrolyzed TEOS adsorbed onto the superparamagnetic MNP surface, which results in transfer of the superparamagnetic MNPs into the water phase [46]. Finally, the hydrolyzed TEOS on the superparamagnetic MNP surface undergoes a condensation reaction to form the silica shell [46]. As the TEOS content increased from 10 to 100 µL, the diameter of MNPs@SiO 2 and thickness of silica shell on the surface of MNP increased. It reached a plateau at 100 µL of TEOS in Figure 4. The silica layer was negligible at 10 µL, while the silica layer was not uniform at 50 µL of TEOS. The MNP@SiO 2 with uniform thickness of silica layer on the surface was obtained at 100 µL of TEOS. The MNP@SiO 2 diameter increased to 24.2 ± 2.0, 31.1 ± 2.3, and 37.7 ± 1.4 nm with 10, 50, and 100 µL of TEOS, respectively (Figure 4b). Increasing the volume of TEOS further caused the formation of bare SiO 2 NPs, while the diameter of MNP@SiO 2 was insignificantly different (Figure 4b,c). So, 100 µL of TEOS was chosen for further study.

Preparation of Graphene Oxide Grafted Polyethyleneimine (GO-grafted PEI)
GO is utilized as a platform structure for conjugating to GPTS-coated MNP@SiO2 in our study. However, GO was easily aggregated in aqueous solution. Therefore, GO was dispersed in solutions of PEI in our study to improve it stability aqueous solution [49][50][51]. GO was incubated with PEI to enables dispersion of GO in aqueous solutions due to its intrinsic hydrophilicity [52]. Therefore, two sizes of branched PEI (molecular weights of 600 (PEI 600), and 25,000 (PEI 25k) were investigated for GO grafting. In Figure S3, GO was dispersed well in PEI 25k but aggregated in PEI 600, as seen in the optical images. This suggests that the low molecular weight of PEI 600 cannot stabilize and dispersed GO in aqueous solutions. TEM images and the absorbance spectra of PEI 25k grafted GO are shown in Figure S3. PEI grafted GO displays a smoother and flatter surface than bare GO. The absorbance spectra of PEI grafted GO shows a significant increase in absorbance intensity in the UV region ( Figure S3), indicating the presence of PEI on the surface of GO.
To increase the linking chain position on the surface, GO was first conjugated with PEI to modify the GO surface with NH2 groups due to its excellent cationic polymer [53][54][55]. To confirm that PEI 25k is covalently bound to GO, we used attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) to characterize the functional groups of PEI grafted GO. FTIR spectra of GO, and PEI 25k grafted GO, are shown in Figure S3. The presence of various oxygen functionalities is present in the GO sample. A broadband and high absorbance intensity from 3000-3700 cm −1 was assigned to the stretching of the hydroxyl group (-OH) [56]. Narrow intense peaks at 1715 cm −1 and 1621 cm −1 represent characteristic stretching of -COO-groups and C=C vibration of aromatic chains on the GO surface. GO also showed peaks at 1352, 1216, 1036, and 974 cm −1 representing the carboxyl/carbonyl, epoxy, and alkoxy groups, respectively [14,[56][57][58].
In contrast, PEI grafted GO exhibited additional peaks at 3100, 2928, and 2821 cm −1 , which are attributed to the stretching of amide B and stretching and bending of -CH2 from PEI. In addition, the

Effect of GPTS Concentration on Silica-Coated Magnetic Fe 3 O 4 Nanoparticles
To conjugate the MNPs@SiO 2 onto the GO-grafted PEI, superparamagnetic MNPs@SiO 2 must be modified with GPTS to create GPTS-coated MNPs@SiO 2 that displayed epoxy groups on the surface, enabling conjugation with the -NH 2 groups of PEIs. The superparamagnetic MNPs@SiO 2 were incubated in a toluene solution containing 1 to 3% GPTS for 1 h. The diameter remained unchanged and no bare SiO 2 was obtained after GPTS treatment ( Figure S2). So, 3% GPTS was chosen for further study.

Preparation of Graphene Oxide Grafted Polyethyleneimine (GO-grafted PEI)
GO is utilized as a platform structure for conjugating to GPTS-coated MNP@SiO 2 in our study. However, GO was easily aggregated in aqueous solution. Therefore, GO was dispersed in solutions of PEI in our study to improve it stability aqueous solution [49][50][51]. GO was incubated with PEI to enables dispersion of GO in aqueous solutions due to its intrinsic hydrophilicity [52]. Therefore, two sizes of branched PEI (molecular weights of 600 (PEI 600), and 25,000 (PEI 25k) were investigated for GO grafting. In Figure S3, GO was dispersed well in PEI 25k but aggregated in PEI 600, as seen in the optical images. This suggests that the low molecular weight of PEI 600 cannot stabilize and dispersed GO in aqueous solutions. TEM images and the absorbance spectra of PEI 25k grafted GO are shown in Figure S3. PEI grafted GO displays a smoother and flatter surface than bare GO. The absorbance spectra of PEI grafted GO shows a significant increase in absorbance intensity in the UV region ( Figure S3), indicating the presence of PEI on the surface of GO.
To increase the linking chain position on the surface, GO was first conjugated with PEI to modify the GO surface with -NH 2 groups due to its excellent cationic polymer [53][54][55]. To confirm that PEI 25k is covalently bound to GO, we used attenuated total reflection-Fourier transform infrared spectroscopy Nanomaterials 2020, 10, 117 9 of 14 (ATR-FTIR) to characterize the functional groups of PEI grafted GO. FTIR spectra of GO, and PEI 25k grafted GO, are shown in Figure S3. The presence of various oxygen functionalities is present in the GO sample. A broadband and high absorbance intensity from 3000-3700 cm −1 was assigned to the stretching of the hydroxyl group (-OH) [56]. Narrow intense peaks at 1715 cm −1 and 1621 cm −1 represent characteristic stretching of -COOgroups and C=C vibration of aromatic chains on the GO surface. GO also showed peaks at 1352, 1216, 1036, and 974 cm −1 representing the carboxyl/carbonyl, epoxy, and alkoxy groups, respectively [14,[56][57][58].
In contrast, PEI grafted GO exhibited additional peaks at 3100, 2928, and 2821 cm −1 , which are attributed to the stretching of amide B and stretching and bending of -CH 2 from PEI. In addition, the disappearance of the 1715 cm −1 peak and occurrence of new peaks at 1640 cm −1 (Amide I) and 1544 cm −1 (Amide II) indicate that the carboxyl groups reacted with the primary amine groups to generate the amide bonds. This result indicates that the conjugation of PEI to the GO surface was successful.
Nanomaterials 2020, 10, x FOR PEER REVIEW 9 of 14 disappearance of the 1715 cm −1 peak and occurrence of new peaks at 1640 cm −1 (Amide I) and 1544 cm −1 (Amide II) indicate that the carboxyl groups reacted with the primary amine groups to generate the amide bonds. This result indicates that the conjugation of PEI to the GO surface was successful.

Conjugation of Silica-Coated Magnetic Nanoparticles on Graphene Oxide Grafted Polyethyleneimine
PEI grafted GO was reacted with GPTS-coated MNPs@SiO2 by incubating 250 µg of GPTS-coated MNPs@SiO2 with PEI grafted GO (250 to 2500 µg) at weight-to-weight ratios (GO/MNP) of 2:1, 3:1, 4:1, 5:1, 6:1, 8:1, and 10:1 (Figure 5a and Figure S4). At a low GO/MNP ratio, GPTS-coated MNPs@SiO2 were aggregated on the GO surface and precipitated. At the highest GO/MNP ratio (10:1), the density of MNPs on the GO surface was too low to impart magnetic properties. Overall, the surface density of GPTS-coated MNPs@SiO2 decreased with increasing GO content. Therefore, a GO/MNP ratio of 5:1 was chosen for subsequent studies. The conjugation of GPTS-coated MNPs@SiO2 onto PEI-grafted GO was confirmed by determining the functional groups with ATR-FTIR (Figure 5b). The GPTS-coated MNPs@SiO2 showed a typical spectrum with peaks at 949 cm −1 and 1053 cm −1 that are attributed to the oxirane group from the GPTS and Si-O from the silica [59]. The intensity ratio of peaks at 949 to 1053 cm −1 is 1.67. When GPTS-coated MNPs@SiO2 were conjugated to PEI grafted GO, the peak ratio between 949 to 1053 cm −1 decreased to 1.05. This result indicates that the oxirane peak of GPTS-coated MNPs@SiO2 The conjugation of GPTS-coated MNPs@SiO 2 onto PEI-grafted GO was confirmed by determining the functional groups with ATR-FTIR (Figure 5b). The GPTS-coated MNPs@SiO 2 showed a typical spectrum with peaks at 949 cm −1 and 1053 cm −1 that are attributed to the oxirane group from the GPTS and Si-O from the silica [59]. The intensity ratio of peaks at 949 to 1053 cm −1 is 1.67. When GPTS-coated MNPs@SiO 2 were conjugated to PEI grafted GO, the peak ratio between 949 to 1053 cm −1 decreased to 1.05. This result indicates that the oxirane peak of GPTS-coated MNPs@SiO 2 reacted with the primary amine groups of PEI. The optical properties of GO/MNP@SiO 2 nanomaterial was showed in Figure 5c. The UV-Vis spectra of GO/MNP@SiO 2 showed the combination properties of both GPTS-coated MNP@SiO 2 with the strong absorbance at~210, 250 and 350 nm and PEI grafted GO at 280 nm. Therefore, GPTS-coated MNPs@SiO 2 were successfully conjugated onto the PEI-grafted GO to generate the GO/MNPs@SiO 2 .

The Magnetization and Protein Isolation of Silica-Coated Magnetic Nanoparticles on Graphene Oxide Grafted Polyethyleneimine
The magnetic property of the GO/MNP@SiO 2 was investigated by using a PPMS-9 vibrating sample magnetometer. The saturation magnetization of the GO/MNPs@SiO 2 was determined to be 9.53 emu/g at magnetic field of 2.0 (T), which is higher than the magnetization of MNPs in previous reports [40,60] (Figure 6a). The magnetization as the function of applied field showed a reversible S-shape and no remnant magnetization is obtained, indicating a superparamagnetic behavior of the GO/MNP@SiO 2 . The magnetic property of the GO/MNPs@SiO 2 was dramatically improved compared to that of MNP in the inset of Figure 6a. After applying of magnet for 5 min, the GO/MNPs@SiO 2 was easily collected, while the MNP@SiO 2 still dispersed in aqueous solution. It indicated that the presence of GO as the platform for conjugating the MNP@SiO 2 facility the collection of MNP.
compared to that of MNP in the inset of Figure 6a. After applying of magnet for 5 min, the GO/MNPs@SiO2 was easily collected, while the MNP@SiO2 still dispersed in aqueous solution. It indicated that the presence of GO as the platform for conjugating the MNP@SiO2 facility the collection of MNP.
For application, GO has been shown to be a suitable platform for protein and nucleic acid adsorption [14,61]. However, the separation of GO from solution is a time-consuming and complicated process [14]. As mentioned above, the MNPs@SiO2 was easily collected when conjugated on the surface of GO in our study, Therefore, we used the GO/MNPs@SiO2 nanocomposites with superparamagnetic property as an isolation material to investigate the isolation of protein in solution. BSA is used as a model protein to investigate the isolation ability of the GO/MNPs@SiO2 nanocomposites. The GPTS-coated MNP@SiO2 was utilized as a control material in our study. As shown in Figure S5, BSA solution exhibited an absorbance peak at 280 nm because of its protein property. This peak was linearly proportional to the BSA concentration in the range of 0 to 100 µM (Figure 6b). The quantity of BSA that adsorbed onto the nanomaterials in our study was observed according to the incubation time. According to the previous report, GO is an amphiphile with both hydrophilic edges and hydrophobic basal plane [62]. The π-π stacking interaction between tyrosine or tryptophan residues of BSA and hydrophobic basal plane have an important role for BSA absorbed on GO/MNPs@SiO2 [62]. Indeed, BSA adsorbed on 1 mg of GO/MNP@SiO2 and GPTS-coated MNP@SiO2 initially increased sharply and achieved a maximum value in Figure 6b. The capability of adsorbed BSA per mg of GO/MNP@SiO2 was 628 ± 9.3 µg/mg after 15 min incubation, adsorbing 65%-69% of the BSA from solution (Figure 6b). The BSA adsorption capability of GPTS-coated MNP@SiO2 was 178 ± 154 µg/mg, which was 3.5-fold less than that of the GO/MNP@SiO2. This result indicates that BSA can be easily adsorbed onto the GO/MNP@SiO2 nanocomposite and collected due to the presence of GO as a platform and MNP as a magnetic separator material. In our previous report, we used graphene oxide conjugated magnetic beads to extract total RNA and miRNA from non-small cell lung cancer cells [14]. Therefore, we believed that it is also possible to isolate RNA and miRNA from a matrix containing sugars, DNA and other biomolecules by using GO/MNP@SiO2 nanomaterial. In conclusion, this result suggests that the GO/MNP@SiO2 nanostructure can be used for protein and nucleic isolation.  For application, GO has been shown to be a suitable platform for protein and nucleic acid adsorption [14,61]. However, the separation of GO from solution is a time-consuming and complicated process [14]. As mentioned above, the MNPs@SiO 2 was easily collected when conjugated on the surface of GO in our study, Therefore, we used the GO/MNPs@SiO 2 nanocomposites with superparamagnetic property as an isolation material to investigate the isolation of protein in solution. BSA is used as a model protein to investigate the isolation ability of the GO/MNPs@SiO 2 nanocomposites. The GPTS-coated MNP@SiO 2 was utilized as a control material in our study. As shown in Figure S5, BSA solution exhibited an absorbance peak at 280 nm because of its protein property. This peak was linearly proportional to the BSA concentration in the range of 0 to 100 µM (Figure 6b). The quantity of BSA that adsorbed onto the nanomaterials in our study was observed according to the incubation time. According to the previous report, GO is an amphiphile with both hydrophilic edges and hydrophobic basal plane [62]. The π-π stacking interaction between tyrosine or tryptophan residues of BSA and hydrophobic basal plane have an important role for BSA absorbed on GO/MNPs@SiO 2 [62]. Indeed, BSA adsorbed on 1 mg of GO/MNP@SiO 2 and GPTS-coated MNP@SiO 2 initially increased sharply and achieved a maximum value in Figure 6b. The capability of adsorbed BSA per mg of GO/MNP@SiO 2 was 628 ± 9.3 µg/mg after 15 min incubation, adsorbing 65%-69% of the BSA from solution (Figure 6b). The BSA adsorption capability of GPTS-coated MNP@SiO 2 was 178 ± 154 µg/mg, which was 3.5-fold less than that of the GO/MNP@SiO 2 . This result indicates that BSA can be easily adsorbed onto the GO/MNP@SiO 2 nanocomposite and collected due to the presence of GO as a platform and MNP as a magnetic separator material. In our previous report, we used graphene oxide conjugated magnetic beads to extract total RNA and miRNA from non-small cell lung cancer cells [14]. Therefore, we believed that it is also possible to isolate RNA and miRNA from a matrix containing sugars, DNA and other biomolecules by using GO/MNP@SiO 2 nanomaterial. In conclusion, this result suggests that the GO/MNP@SiO 2 nanostructure can be used for protein and nucleic isolation.

Conclusions
The silica-coated magnetic iron oxide nanoparticles conjugated to graphene oxide for protein separation were successfully prepared. The silica shell on the superparamagnetic MNPs was optimized by adjusting experimental conditions (300 µL of Igepal ® CO-520, 5 mg of MNP, 100 µL of TEOS, and 100 µL of NH 4 OH in cyclohexane) to produce MNPs@SiO 2 with highly saturated magnetization. MNPs@SiO 2 were incubated with GPTS to produce GPTS-coated MNPs@SiO 2 with surface functionalized epoxy groups. GO was incubated and covalently conjugated with PEI to enhance the stability of GO in aqueous solution and introduce reactive functional groups. GPTS-coated MNPs@SiO 2 were conjugated onto the surface of GO-grafted PEI at a GO/MNP ratio of 5. The saturation superparamagnetization value of the GO/MNP@SiO 2 nanocomposite was 9.53 emu/g at a magnetic field of 2.0 (T). The GO/MNP@SiO 2 nanocomposite was used to separate BSA from solution. The quantity of BSA adsorbed onto 1 mg of GO/MNP@SiO 2 increased sharply and achieved a maximum value of 65%-69% after 15 min. This result suggests that the preparation method for GO/MNP@SiO 2 can be used to fabricate nanocomposites for protein isolation.