Size-Controllable Magnetic Iron Oxide Nanorods for Biomarker

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Size-Controllable Magnetic Iron Oxide Nanorods for Biomarker Targeting and Improving Microfluidic Mixing Yaolin Xu, Hui Wu, Qirong Xiong, Bing Ji, Hong Yi, Hongwei Duan, and Hui Mao ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00359 • Publication Date (Web): 01 Jul 2019 Downloaded from pubs.acs.org on July 18, 2019

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ACS Applied Bio Materials

Size-Controllable Magnetic Iron Oxide Nanorods for Biomarker Targeting and Improving Microfluidic Mixing Yaolin Xu1, Hui Wu1, Qirong Xiong2, Bing Ji1, Hong Yi3, Hongwei Duan2 and Hui Mao*, 1

1Department

of Radiology and Imaging Sciences, Emory University School of Medicine,

Atlanta, Georgia 30322, United States of America 2School

of Chemical and Biomedical Engineering, Nanyang Technological University, 70

Nanyang Drive, Singapore 637457, Singapore 3Robert

P. Apkarian Integrated Electron Microscopy Core, Emory University, Atlanta, Georgia

30322, United States of America

1 ACS Paragon Plus Environment

ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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KEYWORDS: Magnetic nanoparticles; Nanorods; Immunomagnetic separation; Microfluidics; In vitro diagnostics.


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ABSTRACT: Anisotropic nanoparticles, especially gold (Au) nanocubes and nanorods, exhibit unique physical and biological properties compared to their spherical-shaped counterparts, attracting increased attentions and efforts in developing such a class of nanomaterials for enhanced biomedical applications. Here, we report the dimension-controlled preparation of aqueously stable iron oxide nanorods (IONRs) with tunable dimensions (lengths ranging from 25 to 85 nm and diameters from 5 to 16 nm) and varied saturation magnetization values (from 50 to 79 emu·g-1). Subsequently, the prepared IONRs were evaluated for cell uptake and tested for different biomedical applications that can take advantage of strong magnetic properties of IONRs. In immunomagnetic capturing of biofluidic biomarkers, transferrin-conjugated IONRs demonstrated substantial improvement in efficiency (88%) of capturing transferrin receptor overexpressed pediatric brain tumor medulloblastoma cells (D556) comparing to that (47.5%) of commonly used commercial magnetic separation agents, micron-sized Dynabeads®; In detecting Aβs and tau proteins, known as Alzheimer’s disease biomarkers, antibody-conjugated IONRs showed high sensitivity (91.3%) and specificity (88%); Prepared IONRs also demonstrated rotational movement under the controllable alternating magnetic field. By varying the strength and frequency of alternating magnetic field, IONRs can be driven as nano-scaled “stirring bars” in the fluid sample in the biofluidic chamber, leading to enhanced liquid mixing for rapid magnetic separations (completed within 106 seconds).



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1. INTRODUCTION Iron oxide nanoparticles (IONPs) are widely used magnetic materials for developing molecular imaging probes,1 in vitro diagnostic agents,2 image-guided drug delivery,3 and hyperthermia treatments4. In particular, isotropic forms or spherical-shaped IONPs received most attention with the well-documented scalable chemical synthesis5 and a broad range of biomedical applications6. Anisotropic nanoparticles, especially gold (Au) nanocubes and nanorods, have been extensively studied for their shape-specific enhancement in physical and chemical properties and biological functions, which led to increasing interests in developing and investigating shape-controlled nanoparticles. More recently, synthesis and properties of iron oxide nanorods (IONRs) have been reported, demonstrating potentials in biomedical applications.7,

8

Comparing with spherical IONPs, magnetic IONRs have been shown to have

much higher relaxivities enhancing magnetic resonance imaging (MRI) contrast.9, 10 In in vivo applications, anisotropic nanoparticles exhibit favorable pharmacokinetics,11 higher cell uptake and intracellular internalization, and improved tumor targeting and intratumoral retention time12. In in vitro applications, IONRs with much stronger magnetism enable fast and efficient immunomagnetic separation, out-performing even micron-sized magnetic beads.9, 13, 14 Magnetic nanochains made from the linear assemble of spherical IONPs can rotate under the control of an external magnetic field to function as nano-sized stirring bars to enhance liquid mixing in the microfluidic device designed for fast and robust detection of the trace amount of disease specific analytes.15 The integration of targeted capturing capability and magnetic stirring function in magnetic nanochains provides a unique and effective solution in improving microfluidics and sensitivity needed in developing microchips.


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Although these aforementioned advantages of anisotropic nanostructures, the preparation of anisotropic IONPs with controllable aspect ratios remains to be challenging with scarce reports so far.8-10 In early attempts, several strategies were used for the synthesis of anisotropic IONPs or IONRs, including the thermal decomposition of the iron-oleate complex with a carefully-chosen temperature to prepare iron oxide nanowhiskers,16 a seed-mediated route for synthesizing IONRs,17 a surfactant-guided procedure to make iron oxide nanoworms,18 or microwaveassisted19 methods. However, these methods often used highly toxic precursor materials and harsh synthesis conditions. The underlying mechanism for these synthesis method is also unclear and complicated, leading to poor reproducibility and crystallinity of anisotropic IONPs, and limited water dispersity.9 Robust and facile methods to produce IONRs with well-controlled aspect ratios have yet to be established.8, 10 Further understanding the magnetic properties of such a class of nanomaterials is needed in order to develop their magnetism-driven biomedical applications. In this article, we reported a robust route for the synthesis and surface functionalization of highly-magnetic IONRs with tunable dimensions (i.e., 85 x 16, 60 x 10, 40 x 7, and 25 x 5, in length (nm) x diameter (nm)). Three biomedical applications were then used to evaluate the prepared IONRs for their magnetic property-driven performance. For both immunomagnetic cell capturing and separation, and detection of Alzheimer’s disease (AD) biomarkers (i.e., amyloid-β peptides and tau proteins), ligand-conjugated IONRs (60 nm x 10 nm) demonstrated capabilities of capturing tumor cells in the blood sample with high efficiency (88%) and detecting amyloid-β peptides with high sensitivity (91.3%) and specificity (88%), respectively. Furthermore, the reported IONRs showed a 79% increase in cell capturing efficiency over that of widely used commercial micron-sized magnetic Dynabeads®. In addition, IONRs with the dimensions of 85



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nm x 16 nm exhibited excellent magnetic response to an external alternating magnetic field with rotational movement, acting as nano-sized “stirring-bars” in the mixing chamber of a microfluidic device for enhanced liquid mixing and rapid collection into the detection chamber of the device under the external magnetic field.

2. EXPERIMENTAL SECTION 2.1. Materials and chemicals. All chemicals were used without further purification. Ferric chloride hexahydrate (FeCl3·6H2O, 98%), sodium oleate (NaOA), polyethyleneimine (PEI, 50%, 50 to 100 kDa), oleic acid (OA), oleylamine (OL, 70%), 1-octadecene (99.8%), hexane, ethanol (100%), chloroform, dimethylformamide (DMF, 99%), D-(+)-glucose, dimethyl sulfoxide (DMSO, 90%), ammonium hydroxide (NH4OH, ACS grade), sodium bicarbonate (NaHCO3) buffer

(0.1

M,

hydroxysuccinimide

pH=8.5),

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

(Sulfo-NHS),

4′,6-diamidino-2-phenylindole

(DAPI),

(EDC),

N-

fluorescein

isothiocyanate (FITC), paraformalin, phosphate-buffered saline (PBS), fetal bovine serum (FBS), Transferrin (Tf), Eagle's Minimum Essential Medium (EMEM), trypsin-EDTA, cell dissociation solution, and penicillin-streptomycin solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). Amyloid-β protein fragment (1-40), anti-amyloid-β (22-35) antibody, tau protein (tau-441, human recombinant), anti-tau-441 antibody, insulin (human recombinant), bovine serum albumin (BSA), amyloid-β peptide (1-42) (human), and Amicon Ultra-4 centrifugal filters (50 kDa) were purchased from EMD Millipore (Burlington, MA, USA). Anti-amyloid-β (1-28) antibody was purchased from Abbiotec (San Diego, CA, USA). Dynabeads® M-450 Epoxy and the micro BCA protein assay kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Activation and coupling buffer solutions for EDC-NHS coupling reactions were


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purchased from Ocean NanoTech (San Diego, CA, USA). Brain tumor medulloblastoma cell line (D556), lung cancer cell line (A549), breast cancer cell line (4T1), liver carcinoma cell line (HepG2), pancreas carcinoma cell line (MIAPaCa2), cervical cancer cell line (Hela), macrophages (Raw264.7) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). 2.2. Preparation of water-soluble IONRs and spherical IONPs. 2.2.1. Synthesis of β-FeOOH nanorods. The β-FeOOH nanorods were first prepared by hydrolysis of aqueous FeCl3 solution, and then used as the precursor materials for synthesizing IONRs.10 To prepare β-FeOOH nanorods with different lengths (100 x 16, 75 x 10, 50 x 7, or 30 x 5, in length (nm) x diameter (nm)), FeCl3·6H2O (20 mmol) in 100 mL deionized H2O (DI H2O) was mixed with different amounts of PEI (0.1, 0.2, 0.4, or 0.8 mL, respectively) under magnetic stirring. After the mixture was reacted at 80 ºC for 2 hours, β-FeOOH nanorods were collected by centrifugation, and then washed with pure ethanol three times. The residual solution was removed by an overnight drying process within an oven (80 ºC). The resulting β-FeOOH nanorods were in the orange-colored powder form. 2.2.2. Synthesis of IONRs. In a typical synthesis of IONRs, a mixture of β-FeOOH nanorods (307 mg), oleic acid (15 mmol) and oleylamine (15 mmol) was heated to 220 ºC, which was kept for 3 hours under argon gas protection. After the reaction was cooled down, the product was separated out using an EasySep® magnet, washed with a mixture of hexane and ethanol three times, and then dispersed into chloroform as stocking solution (1 mg IONRs/mL) for further transmission electron microscopy (TEM) examination and surface functionalization. IONRs with different lengths were synthesized using the starting materials β-FeOOH nanorods with different lengths.



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2.2.3. Synthesis of spherical IONPs. The hydrophobic spherical IONPs were prepared following our previously published thermo-decomposition method.20 In brief, the iron-oleate complex was first prepared by reacting FeCl3 (4.04 g) with sodium oleate (9.13 g) in a mixture of hexane (50 mL), ethanol (10 mL) and water (40 mL) for 4 hours at room temperature. After the reaction remained still overnight, the upper-layer organic-phase solution containing iron-oleate complex was separated for the synthesis of spherical IONPs. Subsequently, the mixture of ironoleate complex (5 mL) and 1-octadecene (5 mL) was degassed with argon gas for 10 min at room temperature, and heated up to 320 ºC (0.6 ºC/s) for 15 and 30 min to synthesize IONPs with the core sizes of 10 nm or 20 nm, respectively. The resulting IONPs were cleaned with hexane/ethanol solvent mixture (v/v = 1:1) and dispersed into chloroform (1 mg IONPs/mL) before the following surface functionalization. 2.2.4. Water solubilization of IONRs and IONPs with oligosaccharide coatings. To make the prepared IONRs water soluble for further surface functionalization and biological applications, oligosaccharide was introduced onto the surfaces of IONRs to make a hydrophilic coating following our previously reported method of in-situ polymerization of glucose.20 In brief, 1 mL of IONR stock solution was added dropwise into pre-heated DMF (15 mL) solvent with glucose (3 g) dissolved, and the mixture was then gradually (5 ºC/min) heated to 120 ºC and remained under this temperature with magnetic stirring for 2.5 hours. Subsequently, the products were precipitated out and washed by pure ethanol, and re-dispersed into deionized water (1 mg Fe/mL). Iron concentrations were determined with a previously-reported phenanthroline colorimetric method.20 The preparation of oligosaccharide-coated spherical IONPs followed the same aforementioned procedure.


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2.3. Surface functionalization of IONRs. IONRs were first partially ammoniated before conjugating with the green fluorescent dye (FITC, excitation/emission wavelength at 488/525 nm) or FITC-labeled Tf. Fluorescent dyes were introduced onto IONR surfaces to enable fluorescence imaging analysis of IONR attached cells. Tf was selected as the targeting ligand for the magnetic capturing and separation of transferrin receptor (TfR) overexpressed cancer cells with FITC-Tf-IONRs. Briefly, 1 mL of oligosaccharide-coated IONRs (60 nm x 10 nm, 1 mg Fe/mL) was first reacted with 2 mL of ammonia hydroxide at 37 ºC overnight. Then, the ammoniated IONRs were purified with Amicon Ultra-4 centrifugal filters (50 kDa) to remove any ammonia hydroxide residues before being dispersed into coupling buffer (pH = 8.5) at the concentration of 1 mg Fe/mL.20 Subsequently, the FITC (50 µL, 1 mg/mL in DMSO) was reacted with ammoniated IONRs (1 mL of 1 mg Fe/mL) for 2 hours at room temperature to prepare FITC-IONRs, which were further purified with Amicon Ultra-4 centrifugal filters (50 kDa) into water (1 mg Fe/mL) for the future cell uptake studies. To prepare FITC-Tf-IONRs, the FITC (50 µL, 1 mg/mL in DMSO) was first cross-linked to Tf (1 mg) after 2 hour reaction in 0.1 M sodium bicarbonate buffer at room temperature. The resultant FITC-Tf was purified into activation buffer (pH = 5.5) with Tf concentrations of 1 mg/mL. Later, a mixture of EDC (0.5 mg)/NHS (0.25 mg) was reacted with FITC-Tf solution for 0.5 hour to activate the carboxyl groups on Tf. The conjugation between FITC-Tf (Tf concentrations of 1 mg/mL) and IONRs (1 mL of 1 mg Fe/mL) was then conducted for 2 hours at room temperature. Finally, FITC-TfIONRs were purified in deionized water (1 mg Fe/mL) and used as for cell-targeting samples for immunomagnetic cell separation studies. The commercial micron-sized magnetic beads (Dynabeads®, M-450 epoxy, 4.5 μm in diameter) were used as controls, and the preparation of FITC-Tf-Dynabeads® was included in the supporting information.



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To test the capability of IONRs (60 nm x 10 nm) in capturing AD biomarkers (Aβ40, Aβ42 or tau proteins), IONRs (0.9 mg Fe, 5 x 1011 IONRs) were conjugated with anti-Aβ/tau antibodies (0.1 mg) using the same EDC/NHS coupling method applied for making FITC-Tf conjugated IONRs. Anti-Aβ or tau antibody-conjugated Dynabeads® (4.0 × 107) were prepared as a control. The anti-Aβ or tau conjugated IONRs or Dynabeads® were dispersed in water (1 mL) for further studies. 2.4. Characterizations of IONRs. The sizes and morphology of IONRs with different aspect ratios were examined using TEM (accelerating voltage 120 kV, JEOL JEM-1400, Peabody, MA, USA). The size distribution analysis of IONRs was conducted using ImageJ by measuring 100 IONRs on the TEM images. High-resolution TEM was then applied to image the hollow structures within synthesized IONRs. Malvern Zetasizer Nano ZS (Malvern, Worcs, UK) was used to measure the Z-average hydrodynamic size and surface charges of IONRs before and after surface functionalization. The surface coatings of oleic acid/oleylamine-coated IONRs and oligosaccharide-coated IONRs were confirmed with Fourier transform infrared spectroscopy (FTIR). FTIR spectra were collected using a ThermoFisher Nicolet 6700 FTIR spectrometer (Waltham, MA, USA). The crystal structures of IONRs were evaluated using a Philips X’PertDiffractometer with (Bragg-Brentano-geometry, Cu Kα-rays, U = 45 kV; I = 40 mA). The protein ligand (Tf, anti-Aβs, or anti-tau antibodies) concentration on the particles (IONPs, IONRs, or Dynabeads® surfaces was quantified using a microBCA protein assay kit following vendor’s instructions. A Princeton alternating 65 gradient field magnetometer (AGM, Princeton Measurement Corp, Princeton, NJ, USA) was used to measure the magnetic moments of IONRs with different aspect ratios at different applied magnetic field (M-H) at room temperature.


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2.5. Cell uptake of IONRs. To evaluate and compare the cell uptake of spherical IONPs and IONRs, FITC-labeled spherical IONPs (10 and 20 nm) and FITC-labeled IONRs (40 nm x 7 nm, and 25 nm x 5 nm) were incubated with five different cell lines, including 4T1 breast cancer cells, HepG2 liver carcinoma cells, MIAPaCa2 pancreas carcinoma cells, Hela cervical cancer cells, and Raw264.7 macrophages. The cell uptake of FITC labeled spherical IONPs and IONRs was visualized with fluorescence microscopy. Labelling bare spherical IONPs with FITC was followed the procedures of preparing FITC-IONRs described in 2.3. Subsequently, 2 x 104 cells were seeded in 8-well chamber slides and formed a confluent cell monolayer before the treatment of FITC-labeled IONPs and IONRs. Each cell line was separately treated with equal particle number (1 x 1013) of FITC-spherical IONPs and FITC-IONRs for 4 hours at 37 °C. The nanoparticle-treated cells were then washed with 1x PBS, fixed with 4% paraformaldehyde and stained with 4’,6-diamidino-2-phenylindole (DAPI). The cell uptake of FITC-labeled spherical IONPs and IONRs was visualized by BZ-X710 fluorescence microscope. Furthermore, FITC and DAPI signals from fluorescence images were segmented and their pixel numbers were quantified with ImageJ software (developer: Wayne Rasband). The ratios of pixel numbers of FITC over DAPI were used to quantify the cell uptake of IONPs and IONRs by different cell lines. 2.6. Testing immunomagnetic cell separation. Medulloblastoma D556 cells with a high level of TfR expression were used to test ligand-mediated targeting with lung cancer A549 cells with a low level of TfR expression as a control. Cells were cultured following instructions provided by the vendor. After detachment with the cell dissociation solution, D556 or A549 cells (with 10% FBS containing EMEM medium) were seeded (20,000 cells per well) in 8-well chamber slides for further treatment with IONRs or Dynabeads®. After forming a confluent monolayer, cells were incubated with equal particle number (1 x 106) of FITC-IONRs, FITC-Tf-Dynabeads®, or



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FITC-Tf-IONRs for 1 hour at 37 °C. Three replicas were performed for each experiment. IONR or Dynabeads®-treated D556 or A549 cells were then collected and re-suspended into 400 µL of fresh medium before being placed into an EasySep® magnet. After 45 min of magnetic separation at room temperature, the captured D556 or A549 cells were re-dispersed in PBS and counted using a hemocytometer. Separately, the remaining D556 or A549 cells were fixed with 4% paraformaldehyde and stained with DAPI. The bright field and fluorescence images of D556 or A549 cells along with FITC labeled IONR or Dynabeads® were taken using a BZ-X710 fluorescence microscope. 2.7. Testing biofluid sample-based biomarker detection. We tested the ability of IONRs in targeted detection of AD biomarkers, Aβ40, Aβ42, and tau proteins.21 To quantitatively detect these analytes captured by antibody-conjugated IONRs or Dynabeads®, we used FITC and TRITC (red fluorescent dye, excitation/emission wavelength at 557/576 nm) to label Aβ40, Aβ42, and tau proteins, respectively, followed by optical measurements of their fluorescence intensity change before and after separations by targeted IONRs or Dynabeads® conjugated with antibodies against those targets. Insulin and BSA were selected as the non-targeted controls or interfering molecules for Aβs and tau proteins, respectively, due to their similar molecular weight and chemical structure to Aβ40, Aβ42, and tau proteins. In brief, FITC or TRITC (1 mg/mL in DMSO) were added drop-wise to peptide or protein solution (0.5 mg/mL in 0.1 M sodium bicarbonate buffer) with the ratio (w/w) of dye to peptide/protein ratio as 3:50. Specifically, Aβ40, Aβ42 or tau proteins were labeled with FITC while insulin or BSA was labeled with TRITC. After 2-hour incubation, the dye-labeled Aβ peptides or tau proteins mixed with insulin or BSA were purified by removing excess FITC/TRITC and solvents via dialysis, and then quantified with a micro-BCA protein assay kit. Their fluorescence intensity was


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measured at the peptide or protein concentrations of 0, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10, 20 and 50 µg/mL using a BioTek® Synergy 2 plate-reader (BioTek Instrument, Winooski, VT, USA) to obtain the standard curve of the fluorescence intensity as a function of the peptide or protein concentration. To demonstrate the capability of IONRs in capturing Aβ peptides or tau proteins, we prepared Aβ- or tau protein-spiked (10 µg/mL for each analyte) artificial CSF and human blood as the testing samples, respectively. The levels of these targeting biomarkers were selected based on previous reports which showed that clinically diagnosed AD patients have ~100 pg/mL to >10 ng/mL for Aβs and 10 pg/mL to >1 ng/mL for tau proteins in their CSF samples.22, 23 Briefly, FITC labeled Aβs or tau proteins were first dispersed into 0.5 mL of artificial CSF at the concentration of 10 µg/mL, which was then incubated with oligosaccharide-coated IONRs (0.2 mg Fe/mL), anti-Aβ peptide or tau protein antibody-conjugated IONRs (0.2 mg Fe/mL) or Dynabeads® (1×106 beads, recommended by the vendor) for 2 hours. After 1-hour magnetic separation, both captured Aβ peptides or tau proteins and their residues left in the supernatant were collected for fluorescence intensity measurement using a BioTek® Synergy 2 plate-reader. The separation efficiency by different capturing agents is determined by the ratio of measured fluorescence intensity from the captured Aβ peptides or tau proteins to that summed from both captured and residual Aβ peptides or tau proteins. The same protocol was used for the separation of Aβs or tau proteins in the human blood. To evaluate the targeting specificity of antibody-conjugated IONRs to Aβs or tau proteins, we co-incubated the equal concentrations (10 µg/mL) of FITC labeled Aβs or tau proteins with TRITC labeled insulin or BSA with the antibody conjugated IONRs or Dynabeads®. Specifically, FITC-Aβs were mixed with TRITC-insulin, and FITC-tau protein with TRITC-



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BSA, before introducing any capturing agents into the testing samples of artificial CSF or blood. After immune-magnetic separation, the targeting specificity was calculated based on the percentage of captured Aβ peptides or tau proteins over the total percentage of captured materials (Aβ peptides or tau proteins and their corresponding controls or interfering molecules).21 2.8. Testing magnetic stirring effect of IONRs in a microfluidic chamber. The elongated magnetic nanostructures have been shown to rotate under the alternating magnetic field, thus can function as stirring bars to improve the mixing of the fluidic samples in the microfluidic systems that are designed for microscale reactions of different agents required in many “lab on chip” applications, including the “liquid-biopsy” technologies for biomarker detections. We tested the magnetic stirring functions of our IONRs in the previously-reported microfluidic-chip system (MiChip).15 In brief, the MiChip platform consists of a microfluidic mixing chamber placed in the center of four orthogonal electromagnetic coils, mounted on an inverted fluorescence microscope equipped with a high speed CCD camera. The electromagnetic coils can generate a spinning magnetic field with adjustable frequencies, and the movement of magnetic IONRs in this spinning magnetic field can be tracked with a time-lapse high-speed CCD camera. The experiments were performed by injecting 2 µL of oligosaccharide-coated IONRs solution (85 nm x 16 nm, 1 mg Fe/mL) into the microfluidic chamber of MiChip, actuated by the external electromagnetic coils. The coils were set to generate a constant magnetic field with the strength of 80 Gs, but with a varying frequency (40-400 rpm). The magnetic response behaviors of IONRs to the turned-on (40, 100, 150, 200, 325, and 400 rpm) or turned-off electromagnetic field was recorded with the CCD camera. Finally, IONRs were collected into detection chamber of the microchip using a small neodymium magnet (10 x 10 x 5 mm3). The magnetic flux density


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(B) and magnetic field gradient (dB/dx) generated by the small magnet is estimated to be 300 G and 400 G/cm, respectively. The whole separation process of magnetic IONRs was recorded in a video-format for 106s. To better visualize the magnetic stirring and facilitated mixing of the fluids, 1 µL of fluorescent beads (2 mg/mL, 2 μm in diameter) was injected with 1 µL of IONRs (85 nm x 16 nm, 1 mg Fe/mL) into the microfluidic chamber of the microchip. The movement of fluorescent beads was recorded by the CCD camera on the fluorescence inverted microscope at different magnetic field frequency (0, 40, 100, 150, 200, and 325 rpm).

3. RESULTS AND DISCUSSION 3.1. High magnetism and aspect ratio controllable IONRs. The β-FeOOH nanorods, precursor for IONRs, were first synthesized by hydrolysis of FeCl3 solution in the presence of polyethyleneimine (PEI).10 As shown in Figure 1a-d, uniform and monodispersed β-FeOOH nanorods with well-controlled dimensions with lengths and diameters (nm) of 100 x 16, 75 x 10, 50 x 7, and 30 x 5, giving the aspect ratios of 6.25, 7.5, 7.14, and 6, respectively. These were obtained using varied amount of PEI at 0.1, 0.2, 0.4, and 0.8 mL (50 wt. % aqueous solution), respectively. PEI served as the capping agent for β-FeOOH nanorods, which led to the dimension regulations after its protonated form adsorbing onto the lateral plane (200) of β-FeOOH nanorods.24 The similar phenomenon of controlling the dimensions and aspect ratios of nanoparticles using capping agents was observed in the early report using an organic phased synthesis method,8 suggesting that PEI may play a role in controlling dimensions of β-FeOOH nanorods in the synthesis. Subsequently, the organic-phase IONRs were prepared by reduction of β-FeOOH nanorods in the equal molar amount of oleylamine and oleic acid, where oleic acid and



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oleylamine both acted as solvents and capping agents. As an electron donor, oleylamine caused the reduction, which leads to the formation of IONRs at elevated temperature (220 °C).25 However, with the absence of the capping agent, the reaction would yield IONPs with irregular shapes (Figure S1a-b), which demonstrated the importance of oleic acid and oleylamine on the shape evolution of IONRs. The dimensions of prepared IONRs were comparable to their precursors with the lengths slightly shortened to 85, 60, 40, and 25 nm (standard deviation ≤ 15%), respectively. However, the diameters, uniformity and mono-dispersity of IONRs remained unchanged as those of β-FeOOH nanorod precursors (Figure 1e-h). The size distribution of these dimension-tunable IONRs was listed in Figure S1c-f. As results, the corresponding aspect ratios of IONRs were reduced to 5.3, 6, 5.7, and 5.


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Figure 1. TEM images of (a-d) β-FeOOH nanorods, (e-h) oleic acid/oleylamine-coated IONRs, and (i-l) oligosaccharide-coated IONRs with different sizes and dimensions; The XRD spectrum of oligosaccharide-coated IONRs of 60 x 10 in size (length x diameter in nm) with red lines indicating the XRD reference pattern of magnetite (m); FTIR spectra of oleic acid/oleylaminecoated IONRs (black) and oligosaccharide-coated IONRs (red) of 60 x 10 (length x diameter in nm) (n).

It should be noted that some irregular shaped or sized IONPs were observed among IONRs with dimensions of 25 x 5 nm due to the amorphous nature of their initial β-FeOOH nanorod precursors (30 nm x 5 nm), affecting the dissolution and re-crystallization processes during the reduction of forming IONRs.10 Another interesting observation was the presence of microscopic hollow structures in prepared IONRs (Figure S1g-j). This is likely due to collapse of β-FeOOH nanorod framework by release of water molecules during reduction, which may also contribute to the length decrease of IONRs.26 These hollow structures present interesting potentials for drug loading27 and photoacoustic imaging applications28, which will be fully explored in the future studies. To render the prepared IONRs with water dispersity, oligosaccharide coating was applied using the previously reported in-situ polymerization method.20 Figure 1i-l present TEM images of mono-dispersed oligosaccharide-coated IONRs, which showed no change in the morphology, aspect ratios, and uniformity compared to those of IONRs in the organic solvent. In Figure 1m, the peak positions in the XRD spectrum of oligosaccharide-coated IONRs are in good agreement with the XRD pattern of magnetite (solid red lines in Figure 1m) with broadened peaks due to the small and dispersed crystallites. With the existence of oligosaccharide coatings (sharp peak at



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1653 cm-1 for C=C stretching mode) around IONRs, the characteristic peaks of oleic acid (2925 and 2855 cm-1 for C-H stretching mode, 1459 and 1375 cm-1 for CH2 bending mode, and 1541 cm-1 for C=O stretching mode) and oleylamine (3320 cm-1 for N-H stretching mode and 1574 cm-1 for NH2 scissoring mode) were much weakened (Figure 1n)12, 18. DLS and zeta potential measurements were used to study the hydrodynamic size and stability of oligosaccharide-coated IONRs with different dimensions. As shown in Figure 2a, maximal 8 nm increase of the hydrodynamic size was observed in all IONR samples after surface coated with oligosaccharide, which suggested no IONR aggregation in the aqueous solution. The oligosaccharide-coated IONRs are negatively charged (< –30 mV) as the result of ionized hydroxyl groups of oligosaccharide coatings (Figure 2b).20 The negative zeta potential ensures the colloidal stability of IONRs.29,

30

The magnetic properties of oligosaccharide-coated IONRs were evaluated by

measuring the magnetic moment as the function of the applied magnetic field (M−H) curves (Figure 2c) at room temperature. All IONR samples exhibited the superparamagnetic characteristics evidenced by the lack of coercivity in hysteresis curves. The saturation magnetization (Ms) values of IONRs were gradually decreased from 79 to 50 emu·g-1 with a decrease in the length from 85 to 25 nm. Saturation magnetization values of IONRs were relatively smaller than that of bulk magnetite (92 emu·g-1)10. However, the two longest IONRs (85 nm x 16 nm and 60 nm x 10 nm) still possess the magnetism comparable to that of micronsized magnetic beads, Dynabeads® M-450 (~ 70.6 emu/g31), commonly used for magnetic separation in many biomedical applications. Such high magnetism of IONRs may be attributed to the increased spin disorder layer on the surface of IONRs.


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Figure 2. Size (a) and zeta potential (b) measured by DLS and magnetization measurement (MH curves) measured at room temperature (c) of oligosaccharide-coated IONRs with different dimensions.

3.2. Enhanced cell uptake of rod-shaped IONRs. Anisotropic shape-enhanced cell uptake of nanostructures has been reported with silver,32 gold,33 and upconversion nanoparticles34, although little has been known for IONRs. The enhanced or even shape-controlled cell uptake is of great interests when attempting intracellular delivery of therapeutic or imaging agents and better understanding the interactions of nanomaterials with biological systems. We compared the cell uptake of spherical IONPs (10 and 20 nm) and IONRs (40 nm x 7 nm, and 25 nm x 5 nm) by four different types of cancer cells (i.e., 4T1 breast cancer cells, HepG2 liver carcinoma cancer cells, MIAPaCa2 pancreas carcinoma cancer cells, and Hela cervical cancer cells) and Raw264.7 macrophages using the equal number (2 x 1013) of nanoparticles with different forms. FITC was used to label the different IONPs or IONRs to visualize and quantify the cell uptake of spherical IONPs and IONRs by fluorescence microscopy. From the fluorescence images (Figure S3a-t), relatively higher cell uptake of FITC-IONRs than that of FITC-IONPs was observed, evidenced



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by the brighter and more intense green fluorescence from FITC-IONR treated cells. To quantitatively compare the cell uptake of different IONPs or IONRs, areas with green fluorescent signals (FITC) from nanoparticles and blue fluorescent signals from DAPI stained cells were segmented, followed by measuring the pixel numbers of FITC and DAPI and calculating the pixel number ratios (FITC/DAPI) to obtain the ratios of amounts of IONPs or IONRs against the number of cells. The results showed that IONRs had a 2.89-fold (max) increase in cell uptake comparing to spherical IONPs (Figure S3u). Observed shape-enhanced cell uptake of IONRs is consistent with the observations from other anisotropic nanoparticles, such as gold and silver nanorods. The shape dependent cell uptake is thought to be an intrinsic property of the cells with nanorod-preferred endocytosis mediated by clathrin- and caveolae/lipid raft33 and less cell membrane bending energies to overcome during the endocytosis of nanorods.35 The results of enhanced cell uptake of IONRs when compared with IONPs render possible magnetism-based theranostics and biomedical applications (e.g., targeted MRI probes, intracellular drug delivery and immunomagnetic separations) with IONRs. Evaluations of the cell viability with various concentrations (0-130 μg Fe/mL) of IONRs did not show significant cytotoxicity comparing to IONPs, suggesting that prepared IONRs are biocompatible (Figure S5-S9). The toxicity of IONRs and IONPs was both observed at higher concentration (250 μg Fe/mL) (Figure S5-S9), which is similar to the previous report33. 3.3. Efficient cancer cell targeting and immunomagnetic separation with IONRs. 60 x 10 nm IONRs were selected to evaluate ligand mediated targeting of IONRs to cancer cells in vitro because of their comparable magnetic properties to that of Dynabeads® with Ms values of IONRs and Dynabeads® M-450 at 70.47 and 70.6 emu/g31, respectively, and largest aspect ratio leading to enhanced cell uptake.36 To targeted TfR overexpressed D556 cells, successful conjugation of


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FITC-labeled Tf ligands on IONRs was confirmed by the increased hydrodynamic size (from 68 to 79 nm) and decreased zeta potential value (from -16 to -33 mV) of ammoniated IONRs (Figure S2a-b).20 The number ratios of Tf over IONRs and Dynabeads® were found as 7.4 and 38.1, respectively, measured by the BCA assay. FITC-Tf-IONRs were stable in PBS and FBSsupplemented medium (Figure S2a).14 After the FITC-Tf-IONR or FITC-Tf-Dynabeads® treated cells were magnetically separated, captured D556 cells were examined by bright-field microscopy, fluorescence microscopy and Prussian blue staining for iron. Figure 3 shows microscopic images acquired from the same regions of interest. FITC-Tf-IONRs exhibited the intense cell targeting and higher level of cell uptake, indicated by strong colocalized green fluorescence from FITC and blue staining of iron (Figure 3j and 3k), comparing to FITC-TfDynabeads® (Figure 3f and 3g). We also used TEM to examine the location and cellnanoparticle interactions of both FITC-Tf-IONRs and FITC-Tf-Dynabeads®. As shown in Figure 3l, a large amount of FITC-Tf-IONRs were found attached and aligned along the cell membranes of D556 cells (red dashed line) with some internalized by cells (blue dashed circles). In contrast, very few FITC-Tf-Dynabeads® were anchored to the cell membrane (red dashed circles in Figure 3h). This low capture sensitivity of FITC-Tf-Dynabeads® in comparison to FITC-Tf-IONRs is likely due to the stronger steric hindrance of micron-sized beads to cells and limited ligand to cell interactions.37 The results of this comparison may explain the better magnetic separation performance of nano-sized IONRs than the micron-sized Dynabeads®. Without targeting ligands, FITC-IONRs showed nearly no cell targeting nor cellular uptake, indicated by no detectable fluorescence (Figure 3b) and scarce IONR presence around cells in TEM images (Figure 3d). Both FITC-Tf-IONRs and FITC-Tf-Dynabeads® showed little or no uptake by A549 cells without expression of TfR (Figure S4).



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Figure 3. Images of bright-field (a, e and i), fluorescence microscopy (b, f and j), Prussian blue staining (c, g and k) and TEM (d, h and l) of cells captured by FITC-IONRs, FITC-TfDynabeads®, and FITC-Tf-IONRs. All images were taken from the same regions of interest for direct comparison. Red and blue dashed circles in TEM images represent the presence of IONRs and endosomal- or lysosomal-like organelles, respectively; red dashed line and white arrows represent the cell membrane and membrane-aligning FITC-Tf-IONRs); (m) immuno-magnetic cell separation performance tests: after 1 hour (37 °C) incubation with three different capturing


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agents, the captured cells were magnetically collected with an EasySep® magnet (45 min) at room temperature, and then counted using a hemocytometer. To quantitatively evaluate the cell separation performance, 2 x 104 D556 cells were incubated with an equal number of particles (1 x 106) of FITC-IONRs, FITC-Tf-IONRs and FITC-TfDynabeads® at 37 ºC for 1 hour, respectively. After 45 min magnetic separation with an EasySep® magnet, the captured cells were collected and counted using a hemocytometer. The cell separation performance with different capturing agents, defined as the separation efficiency and calculated as the ratio of the number of captured cells over total cells (sum of cells bring captured and cells remained in suspension), is presented in Figure 3m. Consistence with the observation from microscopic images, measurements of cell separation efficiency indicated that FITC-Tf-IONRs captured 85% of D556 cells comparing to only 22% and 47.5% of cells captured by FITC-NRs and FITC-Tf-Dynabeads®. The cell separation efficiency with FITC-TfIONRs was increased by 78% compared to that of FITC-Tf-Dynabeads®. The improved efficiency can be attributed to the increased number of targeting IONRs bound on the single cell and the higher total number of ligands from the all IONRs than that of micron-sized beads12, 37, which facilitated cell targeting due to extended ligand-target interactions.

3.4. Efficient separation of Aβ peptides and tau proteins from biofluidic samples. In addition to immuno-magnetic separation of targeted cells, developed IONRs were tested for targeted capturing of molecular analytes. Deposition of neurotoxic Aβ peptides and aggregation of microtubule tau proteins in the brain are hallmarks of AD, a neurodegenerative disease that has become a major healthcare burden.21 Currently, the diagnosis and monitoring AD is assisted by positron-emission tomography (PET) imaging of brain using specific radioactive tracers to



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detect and quantify Aβ peptides and tau proteins deposited in the brain. However, PET imaging has limited availability due to its cost, short half-time radioisotopes, required hospital visit and complicated imaging procedure and image analysis. Clinical management of AD needs to have more robust, easy administration and cost-effective routine methods as companion tools for the longitudinal measurement of Aβ peptides and tau proteins. Magnetic separation and biomarker detection technologies have been developed and applied as in vitro “liquid biopsy” tools for detecting Aβ peptides and tau proteins spread in the cerebrospinal fluid (CSF) or blood samples.38, 39 Here, we first assessed the separation efficiency of prepared anti-Aβ-IONRs or anti-tau-IONRs (60 x 10 nm) specifically targeting at FITClabeled Aβs or tau proteins (FITC-Aβ- or FITC-tau proteins) spiked artificial CSF (10 µg/mL per analyte). Additionally, the specificity of antibody-conjugated IONRs was evaluated by mixing FITC-labeled Aβs or tau proteins (10 µg/mL for each analyte) with the equal amount of TRITC-labeled insulin or TRITC-labeled BSA. In addition to serve as non-targeting controls, BSA and insulin can also play a role of interfering materials for evaluating the specificity of the Aβ or tau protein targeted IONRs. For comparison, Aβ peptide or tau protein separation performance using oligosaccharide-coated IONRs and antibody-conjugated Dynabeads® was also examined. The ratios of anti-Aβs antibodies over IONRs or Dynabeads® were found as ~1.4 and ~2.6. Similarly, the ratios of anti-tau antibodies over IONRs or Dynabeads® were quantified as ~1.2 and ~2.8. The increased number of antibodies conjugated to Dynabeads® than IONRs is due to the large micron-sized dimensions of Dynabeads®. In the artificial CSF without the presence interfering proteins, antibody-conjugated IONRs and Dynabeads® demonstrated comparable capability of capturing Aβ peptide or tau protein (90.6% vs 95.8% for Aβ40, 91.3% vs 95.3% for Aβ42, and 89.3% vs 91.1% for tau proteins,


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respectively, Figure 4a-c). When insulin or BSA (10 µg/mL) was added in the artificial CSF, antibody-conjugated IONRs only showed a slight decrease (< 5% drop) in their performance of capturing targeted Aβ40, Aβ42 and tau proteins but also exhibited non-specific adsorption of the interfering molecules. 89.3% of Aβ40 vs 33.0% of insulin was captured by anti-Aβ40-IONRs, 87.2% of Aβ42 vs 36.3% of insulin was captured by anti-Aβ42-IONRs, and 88.0% of tau proteins vs to 12.0% of BSA was captured by anti-tau-IONRs, respectively (Figure 4d-f). Thus, the specificity of Aβ40, Aβ42 and tau proteins with antibody-conjugated IONRs is calculated as 73%, 71%, and 88%. However, the specificity of only 50% (ranging from 47.8% to 51.0%) was obtained using oligosaccharide-coated IONRs without targeting antibody ligands or antibodyconjugated Dynabeads®. Capturing Aβ peptides or tau proteins by oligosaccharide-coated IONRs was mainly due to non-specific absorption of Aβ peptides or tau proteins to the oligosaccharide coatings40, which showed no discrimination to insulin, BSA or Aβ40, Aβ42 and tau proteins. The low specificity of antibody-conjugated Dynabeads® was ascribed to the nonspecific absorption of insulin or BSA onto the large surface of micron-sized beads with relative low ligand density (4.4 x 106 ligand/surface area (cm2)) comparing to IONRs with high ligand density (6.9 x 1010 ligand/surface area (cm2)), impeding the targeted detection of Aβ peptides or tau proteins and subsequent magnetic separation.



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Figure 4. The percentages of Aβs and tau proteins captured by antibody-conjugated IONRs, antibody-conjugated Dynabeads® and IONRs without targeting ligands: (a-c) pure Aβs (10 µg/mL) and tau proteins (10 µg/mL) in the artificial CSF; (d-f) Aβs with the presence of nontargeted insulin (10 µg/mL for each), and tau proteins with the presence of non-targeted BSA (10 µg/mL for each) in the artificial CSF; (g-i) pure Aβs (10 µg/mL) and tau proteins (10 µg/mL) in the human blood.

Comparing with CSF, whole blood contains more interfering materials (e.g. serum proteins, various electrolytes, monocytes, red and white blood cells). After spiking FITC-labeled Aβs and tau proteins (10 µg/mL for each analyte) into the whole blood sample, the sensitivity and specificity of antibody-conjugated IONPs and Dynabeads® in capturing Aβs or the tau protein 26 ACS Paragon Plus Environment

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were evaluated. The capturing capabilities of both capturing agents were reduced to 68.3% vs 54.0% of Aβ40 captured by anti-Aβ40-IONRs and anti-Aβ40-Dynabeads®, 69.5% vs 53.1% of Aβ42 captured by anti-Aβ42-IONRs and anti-Aβ42-Dynabeads®, and 68.0% vs 55.4% of tau proteins captured by anti-tau-IONRs and anti-tau-Dynabeads®, respectively (Figure 4g-i). The reduced detection sensitivity was mainly resulted from the non-specific surface absorption of interfering proteins in blood samples by capturing agents, which blocked the binding sites of antibodies to targeted Aβ peptides or tau proteins.41 Interestingly, the relatively less reduction in capturing efficiency (22.3%) was observed from antibody-conjugated IONRs, than that (42.2%) from antibody-conjugated Dynabeads®, further indicating that the capturing abilities of antibodyconjugated IONRs were susceptible to the presence of interfering materials in the detecting medium but less affected comparing to that of micron-sized beads, which possessed relative large surface areas and low targeting ligand density. Neglectable detection of Aβ peptides or tau proteins was observed from oligosaccharide-coated IONRs, which was possibly due to more severe non-specific interactions between IONRs without targeting ligands and complex contents of interfering substances in the whole blood. 3.5. Nano-scale stir-bar capability for improving liquid mixing. It has been shown that anisotropic magnetic nanoparticles can generate rotational movements under alternating external magnetic fields, enabling improved mixing of liquids when used in the microfluidic systems.15 To test whether IONRs can function as such nano-scale “stir bars” in possible microfluidic applications, IONRs with dimensions of 85 x 16 nm were selected because of their longest length for detectable stirring motions and strongest magnetic properties for rapid magnetic separation. The IONR solution (1 mg Fe/mL) was placed in the microchip15 with a pair of external electromagnetic coils that generated a controllable magnetic field with strength (0.008 T) and



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rotational frequency (40-400 rpm). The rotational flow patterns caused by IONRs in the microchip were monitored with a time-lapse high-speed camera. With the magnetic fields turned on-and-off, IONRs responded immediately with clear localized rotational movement, which drove liquid mixing (Movie S1). By periodically activating the orthogonal electromagnetic coils, the well-aligned IONRs exhibited synchronous rotations at different rotating speeds responding to the variable magnetic field frequency. Figure 5a-d show snapshots of IONR rotation (200 rpm) from the recording of the CCD camera at a given magnetic field frequency. Under this magnetic field, IONRs formed a chain-like structure that can be observed at four different directions. Furthermore, linearly aligned IONRs could undergo a synchronized rotation in response to the changing magnetic field with up to 325 rpm frequency without losing their chainlike microstructures. This instant magnetic-field-driven rotation manner of IONRs with adjustable rotating speeds demonstrated the potentials of applying IONRs as nanoscale stirring bars to promote active liquid mixing in microfluidic devices. Enhanced liquid mixing by IONRs was also visualized by observing stirred fluidic motions with fluorescent beads (2 μm in diameter) co-injected with IONRs int the circular mixing chamber of the microchip. Different magnetic field frequencies (0, 40, 100, 150, 200, and 325 rpm) were applied to study the movement behaviors of fluorescent beads (Movie S2) under the CCD camera-mounted fluorescence inverted microscope. In the presence of rotating IONRs, the idly wandering fluorescent beads demonstrated gradually accelerated movement. As the rotational magnetic field frequency was increased, the movement speed of the fluorescent bead increased accordingly, leading to enhanced liquid mixing.


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Figure 5. Snapshots of the CCD camera capturing (a-d) IONRs rotating with an alternating magnetic field (150 rpm) at different time points; (e-h) rapid separation and collection of IONRs by a small neodymium magnet placed in the liquid chamber at different time points of 106 seconds of recording.

Magnetic separation of captured targets in a timely manner is one of the key prerequisites for the development of efficient microfluidic devices. To demonstrate the magnetic separation efficiency of IONRs in the microchip, we recorded the collection process of IONRs into the detection chamber (Movie S3) under a small neodymium magnet (10 x 10 x 5 mm3) with magnetic flux density of 300 G and magnetic field gradient of 400 G/cm. Figure 5e-h show snapshots of the accumulation of IONRs by the magnet in the chamber at different time points. Rapid separation of IONRs (106s, twice speeded up in the video) demonstrated the strong magnetic properties of IONRs with fast magnetic response, resulting in high separation efficiency. With nano-scale stir-bar functions in a miniaturized low-cost microfluidic platform,



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IONRs could enhance the mixing efficiency between capturing agents and analytes with potential multiplexing detections of a diverse range of targets in the microfluidic systems within a short reaction time but high efficiency.

4. CONCLUSIONS We introduced a robust synthesis method for the preparation of highly-magnetic IONRs with controllable dimensions and tunable saturation magnetization up to 79 emu/g. Comparing to spherical IONPs, developed IONRs showed enhanced cell uptake by various types of cells. Coupled with targeting ligands to specific cells or disease specific biomarkers, ligand conjugated IONRs demonstrated the better performance in immunomagnetic capturing and separation of the targeted cells, peptides and proteins, i.e., Aβ peptides and tau proteins of AD biomarkers, comparing to commonly used commercial magnetic separating agents, Dynabeads®. The results demonstrate the advantages of using IONRs to enhance ligand-target interactions to facilitate targeting and improve the capturing efficiency. Furthermore, magnetic IONRs can respond to the alternating rotational magnetic field with rotational motions in the microfluidic systems, leading to the magnetic stirring effect that can promote the liquid mixing in the microfluidic chamber. This unique property of IONRs combining with demonstrated ligand-mediated immunomagnetic separation and capturing capability can be further explored and developed for microfluidic chipbased biomedical applications.

ACKNOWLEDGEMENTS


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The TEM results of this study were obtained with the support from the Robert P. Apkarian Integrated Electron Microscopy Core (IEMC) at Emory University. The JEOL JEM-1400 120kV TEM used in this study was supported by a Shared Instrumentation Grant from National Institutes of Health (S10 RR025679).

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: xxxxx. Figure S1: TEM images of IONPs synthesized with oleic acid (a) and oleylamine (b) only; Figure S2: DLS data of IONRs-60 x 10 nm before and after conjugation with FITC or FITC-Tf, and their stability tests in PBS and FBS-supplemented cell medium (a); zeta potential measurements of IONRs with length x diameter at 60 x 10 nm before and after conjugation with FITC-Tf (b); Figure S3: Fluorescent images of cell uptake of FITC-spherical IONPs and FITCIONRs by four different cancer cell lines and macrophages (a-t); summarization of normalized cell uptake of FITC-NPs (u); Figure S4: Images of bright-field (a, c and e) and fluorescence microscopy (b, d and f) of A549 cells (with low TfR expressions) captured by FITC-IONRs, FITC-Tf-Dynabeads®, and FITC-Tf-IONRs. The scale bar is 20 µm.; Figure S5: Cell toxicity evaluations of spherical IONPs (10 nm and 20 nm) and IONRs (40 nm x 7 nm, and 25 nm x 5 nm) in the 4T1 breast cancer cells; Figure S6: Cell toxicity evaluations of spherical IONPs (10 nm and 20 nm) and IONRs (40 nm x 7 nm, and 25 nm x 5 nm) in the MiaPaCa2 pancreas carcinoma cancer cells; Figure S7: Cell toxicity evaluations of spherical IONPs (10 nm and 20 nm) and IONRs (40 nm x 7 nm, and 25 nm x 5 nm) in the HepG2 liver carcinoma cancer cells; 31 ACS Paragon Plus Environment


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Figure S8: Cell toxicity evaluations of spherical IONPs (10 nm and 20 nm) and IONRs (40 nm x 7 nm, and 25 nm x 5 nm) in the Raw264.7 cell line; Figure S9: Cell toxicity evaluations of spherical IONPs (10 nm and 20 nm) and IONRs (40 nm x 7 nm, and 25 nm x 5 nm) in the Hela cervical cancer cells; Movie S1: Bright-field imaging of IONRs responding to an alternating on and off magnetic field, captured by a CCD camera at 0.1 s intervals; Movie S2: The active liquid mixing experiments by mixing fluorescent beads and IONRs, captured by a CCD camera at 0.1 s intervals; Movie S3: The magnetic separation of IONRs into the detection chamber of the microchip (the video is 2x speeded up).

AUTHOR INFORMATION Corresponding Author *Hui Mao, PhD, 1Department of Radiology and Imaging Sciences, Emory University, Atlanta, Georgia 30329, USA, Email: [email protected]

Funding Sources This study is supported in parts by NIH grants (R01CA154846-04 and U01CA151810-05 to HM).

Notes The authors declare no competing financial interest.


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Graphical Abstract


Size-Controllable Magnetic Iron Oxide Nanorods for Biomarker

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