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Scalable Solvothermal Synthesis of Superparamagnetic FeO Nanoclusters for Bio-separation and Theragnostic Probes Jeonghyo Kim, Van Tan Tran, Sangjin Oh, Chang-Seok Kim, Jong Chul Hong, SungIl Kim, Young-Seon Joo, Saem Mun, Myoung-Ho Kim, JaeWan Jung, Jiyoung Lee, Yong Seok Kang, Ja-Won Koo, and Jaebeom Lee

ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b14156 • Publication Date (Web): 22 Nov 2018 Downloaded from http://pubs.acs.org on November 24, 2018

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ACS Applied Materials & Interfaces

Scalable Solvothermal Synthesis of Superparamagnetic Fe3O4 Nanoclusters for Bioseparation and Theragnostic Probes Jeonghyo Kim1, †, Van Tan Tran1, †, Sangjin Oh1, †, Chang-Seok Kim1, Jong Chul Hong2, SungIl Kim3, Young-Seon Joo3, Saem Mun3, Myoung-Ho Kim3, Jae-Wan Jung3, Jiyoung Lee4, Yong Seok Kang4, Ja-Won Koo4, Jaebeom Lee5,* 1Departments

of Cogno-Mechatronics Engineering, Pusan National University, Busan,

46241, Republic of Korea 2Department

of Otolaryngology, Head and Neck Surgery, College of Medicine, Dong-A

University, Busan, 49201, Republic of Korea 3AMO

LIFE SCIENCE Co., Ltd., Seoul, 06527, Republic of Korea

4Department

of Otorhinolaryngology-Head and Neck Surgery, Seoul National University

Bundang Hospital, Seongnam, 13620, Republic of Korea 5Department

of Chemistry, Chungnam National University, Daejeon, 34134, Republic of

Korea

†These

authors contributed equally.

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ABSTRACT Magnetic nanoparticles have had a significant impact on a wide range of advanced applications in the academic and industrial fields. In particular, in nanomedicine, the nanoparticles require specific properties, including hydrophilic behavior, uniform and tunable dimensions, and good magnetic properties, which are still challenging to achieve by industrial-scale synthesis. Here, we report a gram-scale synthesis of hydrophilic magnetic nanoclusters based on a one-pot solvothermal system. Using this approach, we achieved the nanoclusters with controlled size composed of magnetite nanocrystals in close-packed superstructures that exhibited hydrophilicity, superparamagnetism, high magnetization, and colloidal stability. The proposed solvothermal method is found to be highly suitable for synthesizing industrial quantities (gramper-batch level) of magnetic spheres with unchanged structural and magnetic properties. Furthermore, coating the magnetic spheres with an additional silica layer provided further stability and specific functionalities favorable for biological applications. Using in vitro and in vivo studies, we successfully demonstrated both positive and negative separation, and the use of the magnetic nanoclusters as a theragnostic nanoprobe. This scalable synthetic procedure is expected to be highly suitable for widespread use in biomedical, energy storage, photonics, and catalysis fields, among others.

KEYWORDS: Magnetic nanoclusters, Scalable synthesis, Superparamagnetism, Magnetic separation, Theragnosis 2 ACS Paragon Plus Environment

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INTRODUCTION Magnetic nanoparticles (MNPs) have attracted attention for applications in biomedicine and therapeutic fields over the past few decades due to their unique properties.1-4 MNPs are one of the most promising tools for isolation and/or enrichment of biological targets. MNPs with affinity tags can capture targets, such as proteins, nucleic acids, or cells via various biological interactions. A magnetic force applied to the target-bound MNPs is exerted along the noncovalent binding interface of the captured targets, which enables controlled guidance of biomolecules in the liquid medium without being impeded by viscous drag and Brownian forces.5 A further exciting research topic is the development of MNPs combined with different functional nanomaterials;6-10 in particular, magnetic-fluorescent particles are promising for advanced applications. Various organic and inorganic luminescent materials, such as commercial dyes11,12 and quantum dots,7 have been successfully used as optically active centers and incorporated with MNPs. MNPs used for the magnetic vehicles in the biological fluids require superparamagnetism and sufficient magnetization per particle. Superparamagnetic iron oxides (SPIOs) are considered ideal candidates for these purposes, however, their use in bio-separation and magnetic targeting treatment has been impeded by low magnetization and small MNP diameters.13 To enhance the magnetic response, efforts have been made to increase the particle size, along with doping stronger magnetic components such as Co and Ni.14 Increasing the particle size increases the saturation magnetization, but an inevitable transition from superparamagnetic to ferromagnetic behavior occurs with increasing particle size, resulting in aggregation of the ferromagnetic particles in fluidic environments. An alternate strategy of replacing the divalent or trivalent Fe ions with cations (e.g., Zn2+, Mn2+, Co2+, and Ni2+) results in increasing both the saturation magnetization (MS) and magneto-crystalline anisotropy, which are advantageous in biological and data storage applications, respectively.15,16 Hence, a novel 3 ACS Paragon Plus Environment


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strategy was proposed to fabricate a microscale cluster from numerous nanoscale particles (~10 nm diameter), thereby greatly increasing the saturated magnetization while retaining individual magnetic domains and superparamagnetic behavior on the microscale.17 Based on this strategy, many recent studies successfully demonstrated the use of magnetic clusters in advanced applications, such as bio-separation, targeted delivery, and imaging.7,18-21 In addition to the magnetization, other capabilities and functionalities of the clustered magnetic nanomaterials can be effectivity enhanced compared with individual nanoparticles, broadening the potential biomedical applications. For example, Fe3O4 magnetic nanoclusters (~40 nm) comprising NPs of ~6 nm exhibited a significant increase in the transverse relaxation and relaxation ratio, which is often observed by enhanced efficacy of the T2 contrast agents in MRI, compared with single NP.22 Furthermore, MNPs of 100–200 nm in diameter have been reported to possess a long circulating plasma half-life for imaging and targeting via enhanced permeability and retention phenomena of the tumor.23,24 Solvo/hydrothermal synthesis methods have been extensively developed as they provide a simple, versatile, and cost-effective route for producing large quantities of highly crystalline magnetic clusters with controlled morphology.23-26 A main advantage of solvothermal synthesis is the ability to tailor the size of both the primary nanocrystals and the secondary clusters, which primarily determines the magnetic behavior of the obtained particles.23 Tang et al. proposed a method for synthesizing sub-100 nm biocompatible Fe3O4 clusters using water as a size-control agent and sodium citrate as a stabilizer.27 However, the presence of water can degrade the particle crystallinity. A water-free synthesis method was used to tune the grain size and cluster size using a bisolvent system.23,24 The average nanograin size and secondary Fe3O4 particles were tuned in the range of ~5.9–21.5 nm and 6–170 nm, respectively, by simply changing the weight ratios of sodium acrylate/sodium acetate the volume ratios of ethylene glycol/diethylene glycol (DEG), respectively. However, the particles suffered from low 4 ACS Paragon Plus Environment

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aqueous dispersibility and low biocompatibility due to a lack of surfactant. Furthermore, DEG is acutely toxic.23 Large-scale synthesis is important in order to reduce manufacturing costs and batch-to-batch variations to allow further clinical use of MNPs; however, successful scale-up of laboratory protocols to an industrial level is often complicated. The current definition of large-scale NP production is usually gram-scale or higher.13 Scalable production of hydrophobic iron oxide NPs based on thermal treatments in the organic phase has been widely studied. A method for synthesizing monodispersed iron oxide NPs via thermal decomposition produced up to 40 g of products in a single batch.28 Synthesis of iron oxide nanocrystals of 20 nm) with high crystallinity via control of the crystal growth process by the pressure–temperature environment;26 (ii) cost-effective single-step process that does not require any seeds, catalysts, toxic and expensive surfactants, or templates;31 (iii) a homogeneous nucleation processes, narrow particle size distribution, and controlled particle morphology can be obtained due to elimination of the calcination step. The ability to precipitate powders directly from solution regulates the rate and uniformity of



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nucleation, growth, and aging, which allows unique control of the size, morphology, and aggregation.32,33 In this study, we investigated a solvothermal synthesis method for the up-scaling of a closepacked superstructure of magnetic nanoclusters (MNCs), in which the morphology of the MNCs was tuned by varying the molar ratios of precursors and surfactants. We evaluated the introduction of a silica layer considering the stability and functionalities required for various biological systems. Silica-coated MNCs (silica-MNCs) were used for bio-separation by magnetically manipulating the movement of various biological substances, and the capture efficiency and target recovery were evaluated. Furthermore, fluorescent-MNCs (Europium doped Fe3O4@SiO2 core-shell structure) were fabricated as multifunctional nanocomposites by introducing rare-earth fluorophore-loaded silica shells. In vivo fluorescence imaging of hair cell in a mouse model of hearing loss was investigated to evaluate the fluorescent-MNCs as a theragnostic reagent, where hair cell damage is related to hearing loss.

RESULTS AND DISCUSSION

Figure 1. Gram-scale synthesis of Fe3O4 nanoclusters. (a) Schematic illustration of the procedure for the gram-scale synthesis of MNCs. (b) Proposed mechanism of the formation of the clustered Fe3O4 nanocrystals structure. (c-e) A set of TEM images of MNCs at different magnifications. (f) SEAD pattern acquired from a single Fe3O4 nanocluster. 6 ACS Paragon Plus Environment

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Gram-scale synthesis of hydrophilic MNCs was performed using a facile one-step solvothermal method by reduction of FeCl3 with EG at 200°C in the presence of sodium acetate as an alkali source and biocompatible trisodium citrate (TSC) dihydrate as a stabilizer (Figure 1a, b). Figure 1c and Figure S1 show typical high-resolution transmission electron microscopy (HR-TEM) and scanning electron microscopy (SEM) images of the as-prepared monodisperse MNCs with a nearly spherical shape and uniform size of c.a. 200 nm. HR-TEM image (Figure 1d) revealed that the MNCs were composed of small primary Fe3O4 nanocrystals with a size of 11–13 nm. The nitrogen adsorption-desorption isotherms of the sample are shown in Figure S2a, indicating type IV isotherm for mesopores.34 The pore size distribution curve determined by the Barrett–Joyner–Halenda (BJH) method indicates that there are peaks in the microporous and mesoporous range, a dominant peak around 1.4 nm and small peaks at 2.27 nm and 3.9 nm (Figure S2b). The micropores and mesopores on the MNCs can be attributed to the interspaces of the constituent nanocrystals. The surface area of the MNCs is 27.543 m2 g−1 which is significantly larger than the value for single crystal magnetite hollow spheres reported in the literature (16.251 m2 g−1).35 The lattice fringes observed in Figure 1e are about 0.483 nm and 0.292, which are consistent with the interplanar distance of (111) and (220) lattice planes. The diffraction rings of the selected-area electron diffraction (SEAD) pattern taken from the corresponding single cluster in Figure 1c suggest that the MNC is polycrystalline. From inside to outside, the rings can be indexed to (111), (220), (311), (400), (511), (440) planes of spinel Fe3O4, respectively (Figure 1f).



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Figure 2. Characterization of Fe3O4 nanoclusters. (a) XRD pattern, (b) XPS spectra, and (c) magnetic hysteresis curve of the MNCs measured at 5 K (black) and 300 K (red). (d) TGA (solid black lines) and DSC (dashed red lines) curves of bare Fe3O4 NPs, and TSC-stabilized MNCs. (e) FT-IR spectrum.

X-ray diffraction (XRD) measurements were used to characterize the structure of the MNCs; the XRD pattern showed six well-resolved diffraction peaks, which were indexed to the (220), (311), (400), (422), (511), and (440) planes of the cubic Fe3O4 phase (International Center for Diffraction Data (ICDD) card No. 19-0629) (Figure 2a). The average nanocrystal size was ~13.8 nm, calculated using the Scherrer formula for the (311) peak; this agreed well with the TEM data. Since magnetite (Fe3O4) and maghemite (γ-Fe2O3) exhibit similar XRD patterns, the composition of the MNCs was further identified by X-ray photoelectron spectroscopy (XPS) because of its sensitivity to Fe2+ and Fe3+ cations.36 Figure 2b shows the representative high-resolution XPS pattern of the MNCs, revealing that the binding energies of Fe 2p3/2 and 8 ACS Paragon Plus Environment

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Fe 2p1/2 are 710 and 724 eV, respectively, close to the published literature about Fe3O4 NPs.37 Moreover, the total absence of a peak at 719.0 eV could further identify that the product is Fe3O4 rather than γ-Fe2O3.38 In a typical solvothermal process, EG acts as both the solvent and reductant at a relatively high boiling point, while NaAc·3H2O was added for two purposes: (i) as an electrostatic stabilization agent to prevent nanocrystal agglomeration; and (ii) as a reduction agent to provide strongly alkaline conditions to promote the hydrolysis of iron ions in solution.39 TSC was chosen as the surfactant due to its strong coordination affinity to Fe3+ ions, which favors the attachment of citrate groups on the surface of the magnetite nanocrystals and prevents them from aggregating into large single crystals.40 The formation of polycrystalline Fe3O4 nanoclusters follows the well-documented two-stage growth model in which primary nanocrystals nucleate first in a supersaturated solution, and then aggregate into larger secondary particles (Figure 1b). Initially, the primary Fe3O4 nanocrystals were formed and stabilized by adsorbed citrate molecules, resulting in electrostatic repulsion between neighboring nanocrystals. However, the high surface energy of the nanocrystals promoted merging of the crystals to reduce it in the solvent.41 The balance of these two opposing forces governs the aggregation of nanoclusters, and hence, the secondary nanosphere size. The magnetic properties of the MNCs were investigated using a SQUID magnetometer at 5 K, and 300 K (Figure 2c). The magnetic hysteresis loop at 300 K shows a saturation magnetization of ~68 emu/g with no remanence and coercivity, indicating superparamagnetic property. Meanwhile, at 5 K, thermal energy is too weak to induce moment randomization, so that the MNCs show typical ferromagnetic hysteresis loops with a remanence of 19 emu/g and a coercivity of 240 Oe (Figure 2c inset). It usually attributed to the grain sizes of the primary nanocrystals being well below the critical size (25–30 nm) of the superparamagnetic– ferromagnetic transition (Figure S3).42 The ferromagnetic behavior of cluster may be attributed 9 ACS Paragon Plus Environment


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either to ferromagnetic incorporated nanocrystals of sufficiently large diameter ( > 20 nm) or too strong intra-cluster interactions (including exchange interactions and dipole-dipole interactions).43,44 Because of small primary Fe3O4 nanocrystals, it is likely that the nanogaps of the organic surfactant between neighboring nanocrystals reduce the intra-cluster interactions, contributing to the absence of the superparamagnetic–ferromagnetic transition in our data (Figure 2c).45 To quantify the amount of surfactant on the surface of Fe3O4 nanoclusters, thermogravimetric analysis/differential scanning calorimetry (TGA/DSC) analyses were performed for both bare and TSC-stabilized MNCs. As shown in Figure 2d, the samples were heated up to 700°C at a rate of 10°C·min-1. The remaining weight percentages of bare and TSC-coated MNCs were 97.76% and 90.21%, respectively. When Fe3O4 nanoclusters were stabilized with a surfactant, typical degradation stages were observed in the TGA curve. The weight loss from 250 to 350°C was attributed to the removal of TSC molecules bound to the Fe3O4 surface. A distinct exothermic peak was observed at ~270°C, which was used to calculate the content of TSC (8.12 wt.% per mg of MNCs). The small reduction above 570°C was related to the conversion of Fe3O4 to γ-Fe2O3 and FeO, which are the stable phases in the Fe-O system. The FT-IR spectra showed further evidence of the formation of TSC-coated MNCs; as shown in Figure 2e, where both characteristic bands from Fe3O4 and TSC were observed. The peaks at 580 cm−1 and ~3000 cm−1 are related to the stretching vibration of Fe–O and surface hydroxyl groups, respectively. The absorption bands located at 1716 cm−1 and 1520 cm−1 were identified as COO– asymmetric stretching of C=O and C–O vibration symmetric stretching from the COOH group, respectively, indicating that the carboxyl groups were strongly coordinated to the surface of Fe3O4 nanoclusters. An extra band was observed at 1156 cm−1 which was attributed to the C–H bending vibration mode of the TSC used during synthesis. Measurement of the zeta potential of the citrate-coated MNCs as a function of pH confirmed the surface charge 10 ACS Paragon Plus Environment

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properties. The pH values of the suspension were adjusted in the range of 2–12 (Figure S4a, b). The surface charge properties were mainly attributed to the terminal carboxyl group that was strongly coordinated with Fe cations at the MNC surface. At low pH (pH < isoelectric point (IEP); IEPMNCs=4.5), the carboxyl groups were protonated, resulting in a positively charged surface. When the solution pH was above the IEP, the carboxyl groups become deprotonated, resulting in negatively charged nanoclusters. The electrostatic repulsive force has an effect over a wide range of pH values (5.5–12), which allows the MNCs to be well dispersed in a variety of buffers and physicochemical environments (Figure S5).

Figure 3. Size tunability of the Fe3O4 nanoclusters. (a–h) Representative SEM and TEM images of the MNCs synthesized with a Fe precursor concentration of (a, e) 0.05 M, (b, f) 0.1 M, (c, g), 0.2 M, and (d, h) 0.3 M. (i) Size distribution of MNCs obtained by measuring 100 particles from TEM images. (j) Calculated average diameters and zeta potentials as a function of Fe precursor concentration. 11 ACS Paragon Plus Environment


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Table 1. Average diameter, relative standard deviation (%RSD), and zeta potential of Fe3O4 nanoclusters synthesized with different concentrations of Fe3+ ion precursor. Concentration of Fe3+ (M)

Diameter (nm)

RSD (%)

Zeta potential (mV)

0.05

117.5 ± 13.8

11.8

-41.4

0.1

213.3 ± 21.2

9.9

-34.6

0.2

281.3 ± 28.4

10.1

-27.7

0.3

328.5 ± 28.5

8.7

-14.5

Here, Fe3O4 MNCs with tunable sizes were obtained by controlling the molar ratio of [FeCl3]/[TSC]. SEM and TEM images of Fe3O4 clusters obtained using FeCl3 concentrations of 0.05–0.3 M showed that all clusters had a spherical shape and uniform size with an average diameter increasing from 117 to 328 nm with increasing FeCl3 concentration (Figure 3a–h, Table 1). This was attributed to a reduction in the electrostatic repulsion between neighboring nanocrystals due to a lower density of citrate molecules on the surface of the magnetite nanocrystals at higher FeCl3 concentrations. Consequently, the magnetite nanocrystals tended to aggregate into larger clusters. The particle uniformity was high comparing different samples (RSD = ~10%) (Figure 3i, Table 1). Figure 3j shows the dependence of the diameter and zeta potential of the clusters on the Fe precursor concentration; with increasing FeCl3 concentration, the negative surface charge reduced from -41.4 to -14.5 mV (Figure S6) due to the lower citrate density.


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Figure 4. Scalable synthesis of magnetic nanoclusters. (a) The mass yield, particle sizes, zeta potentials, and polydispersity index (PDI) values of MNCs obtained by solvothermal reactions with different reaction volumes ([FeCl3] = 0.1 M, [TSC] = 0.034 g/mL, [NaAc] = 0.125 g/mL, reaction temperature = 200°C, and time = 10 h). (b, c, and d) Size distribution of Fe3O4 nanoclusters synthesized with different reaction volumes. The insets are representative SEM images of each sample. (e) XRD patterns, and (f) magnetic hysteresis loops (at 300K) of the MNCs obtained with different reaction volumes. (g) Schematic diagram of the overall mass production procedure for monodispersed Fe3O4 nanoclusters, where lab-scale (~1 g) and pilotscale (200 g) quantities are shown. (h) Representative SEM images showing highly uniform MNCs synthesized using the pilot-scale reactor.



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Table 2. Mass yield, particle size, zeta potential, PDI, estimated grain size, Ms of Fe3O4 nanoclusters synthesized with different reaction volumes. Reaction volume (mL)

Yield rate (mg/hour)

20

15.5 ± 0.5

40

27.6 ± 0.8

200

160.7 ± 2.1

Diameter (nm)/ RSD (%) 208.5 ± 13.5/ 6.5 224.6 ± 24.2/ 10.8 213.7 ± 18.6 /8.7

Zeta potential (mV) -34.0 ± 0.6 -29.4 ± 1.2 -32.4 ± 0.8

PDI 0.153 ± 0.03 0.194 ± 0.08 0.183 ± 0.02

Estimated grain size (nm)

Saturation magnetization (Ms, emu/g )

14.14

66.44

14.93

66.24

13.42

63.34

The mass yield of a single-batch synthesis could be easily scaled up by increasing the reaction volumes while maintaining the same concentration of all chemicals. The product yield, particle size, surface charge, and polydispersity index (PDI) were investigated at various reaction volumes using autoclaves of 20, 40, and 200 mL. The mass yield of MNCs linearly increased from ~155 mg to ~1607 mg, corresponding to the yield rate of 15.5±0.5 to 160.7±2.1 mg/hour as the reaction volume increased from 20 to 200 mL. However, the particle size and polydispersity stayed within a narrow distribution range, and the surface potential was constant (around -30 mV), even when the reaction volume was ten times larger than the lab-scale test (Figure 4a–d, and Table 2). Their crystal structures and grain sizes, and magnetic properties were investigated by X-ray diffraction pattern and hysteresis curve. XRD patterns are basically the same for the samples synthesized at different reaction volumes. The grain sizes calculated from the XRD data using Scherrer formula are almost unchanged for different reaction volumes (Figure 4e and Table 2), revealing that increasing the reaction volume at the levels we have tested does not affect the crystal structure and the grain size of the MNCs. All samples show similar magnetic hysteresis curves with the superparamagnetic feature (no remanence and no


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coercivity) except a slight decrease of saturation magnetization from 66.44 to 63.34 emu/g as the reaction volume increased from 20 to 200 mL (Figure 4f and Table 2). These results verified that the solvothermal synthesis method is viable for industrial-scale production of MNCs. Although the experiments were limited by the availability of larger capacity reactors, we recently developed a mass production reactor capable of synthesizing hundreds of grams of MNCs daily (Figure 4g, h); prototypes of surface-modified MNCs have been commercialized to the market.

Figure 5. Negative or positive separation of biomarkers in crude samples. (a) Overview of the negative and positive separation procedures using immuno-silica MNCs. (b) Red blood cell (RBC) isolation from whole human blood. (Top) Photographs before and after RBC separation, 15 ACS Paragon Plus Environment


and (bottom) optical microscopy images obtained from the supernatant and precipitate. The real-time RBC separation process is shown in Supplementary Movie S1. (c) Western blot analysis of enriched C-reactive protein from human serum. (d) Vibrio bacteria (V. parahaemolyticus, KCCM 11965) enrichment from culture media.

The observed properties of the MNCs, such as water solubility, superparamagnetism, and strong magnetic responses, show their high potential for nano-biomedical applications. To demonstrate the practical usability of the MNCs, we first evaluated the performance of efficient bio-separation of biological targets of various sizes, including red blood cells (RBC) = ~7–8 μm, C-reactive proteins (CRP) = ~10 nm, and Vibrio bacteria = ~1.5 μm (Figure 5a). MNPbased separation offers a method for separating biomarkers in a rapid, efficient, and costeffective manner.1,6,20,46,47 The silanization of the magnetic cores is the best choice to ensure high stability, dispersity, and useful surface functionality1,4, however, slight reduction of saturation magnetization (~14%) was inevitable after coated with the nonmagnetic layer of SiO2 (Figure S7).The silica-MNCs were functionalized with target-specific antibodies using EDC/sulfo-NHS zero-length conjugation, and then added to a suspension containing the target of interest. Following magnetic separation, the capture efficiency was calculated by measuring the amount of target proteins or cells as a percentage of the total number of cells. First, the assay for negative separation was designed for the isolation of rare circulating tumor cells (CTCs) from the blood of patients; this is an important factor for elucidating cancer metastasis and prognosis.48 In general, positive-CTC isolation strategies exploit antibodies against surface antigens, such as the epithelial cell adhesion molecule (EpCAM). However, these methods may not be able to detect tumor cells that have lost EpCAM expression or have a non-epithelial origin; in addition, the expression of specific biomarkers varies greatly depending on the patient, which reduces the efficacy and reliability of the analysis.49 To overcome these limitations, the negative-depletion strategy could be effective as it provides tumor antigen16 ACS Paragon Plus Environment

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independent enrichment by depleting normal existing cells in the blood, such as leukocytes, and erythrocytes.50,51 For example, Toner et al. developed a microfluidic CTC isolation chip using magnetophoresis for depletion of leukocytes, which successfully separated 97% of rare cells with an 8 mL/h processing speed.49 Here, we demonstrated high-efficiency negative isolation of RBCs from whole human blood samples. Briefly, the anti-RBC antibody (Ab)silica-MNCs were added to 25 μL of whole blood. After 5 min of reaction, the blood sample changed from red to a transparent solution as the RBCs were captured and separated by the magnetic particles (Figure 5b (top), and Movie S1). The specific binding of Ab-silica-MNCs (black dots) to the outer membrane of RBCs was visualized by optical microscopy, where neither RBCs nor nanoclusters were observed in the supernatant (Figure 5b (bottom)). The RBC separation efficiency was 99.5%. Thus, this robust and straightforward negative cell depletion method can be applied to the rapid analysis of CTCs in studies of various cancers. Next, we experimentally demonstrated the high-recovery positive separation of CRP and Vibrio bacteria from their crude solution (human serum and culture media, respectively) using the silica-MNCs. CRP is an important prognostic indicator of cardiovascular disease (CVD), and the elevation of its level in serum is associated with recurrent CVD incidence and higher death rates. However, the clinically significant detection range of CRP is extremely low (pM to nM); therefore, highly sensitive quantitative detection of CRP enables early diagnosis of CVD and quick response for patient care.52 In this test, anti-CRP antibody-crosslinked silicaMNCs were mixed with a CRP spiked serum sample (1 μg/100 μL) for 10 min, followed by separation from the serum background. Then, the bound CRPs were released from the magnetic particles using a glycine elution buffer and were analyzed by western blotting (Figure 5c). The western blot signals from these CRPs (‘retrieved’) were compared with a series of diluted CRPs ranging from 10 to 100 ng (‘standard’) which confirmed the enrichment of antigens from the sample matrix. Foodborne illnesses caused by pathogenic bacterial infections are an important 17 ACS Paragon Plus Environment


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public health problem. However, pathogens usually exist in complex food matrices that are highly infectious at low dose exposures; thus, effective enrichment and isolation of pathogens are required for food safety testing.53 Vibrio parahaemolyticus (KCCM 11965) was chosen as a model bacteria, and the anti-Vibrio antibody immobilized silica-MNCs were incubated with 2, 25, and 255 CFU/mL of bacterial suspension for 30 min. After magnetic separation, the captured bacteria were retrieved in a PBS solution for plate counting, and the capture efficiency was calculated. The tested bacterial assay showed a capture efficiency of >99.2% for different concentrations, and non-specific targeting of

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