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  • br Cancer cell separation using Ab MNP Si in


    2.7. Cancer cell separation using Ab/MNP-Si in a mixture of fresh human whole blood
    To analyze the ability of Ab/MNP-Si to isolate HER2+ cells, SK-BR-3 GSK126 were stained with carboxyfluorescein succinimidyl ester (CFSE) as a live cell-staining fluorescent dye (which changes the color of the cells to green) according to the manufacturer’s protocol. Stained SK-BR-3 cells were then mixed with 1 mL fresh human whole blood. A volume of 90 µL Ab/MNP-Si was added to the SK-BR-3/whole blood mixture and stirred at room temperature for 1 h. The mixture was then placed on a magnet for 20 min to allow magnetic isolation of the Ab/MNP-Si attached cells. The captured Ab/MNP-Si were attached on the tube wall, and the supernatant fluid was discarded. The pellet containing 
    Ab/MNP-Si was resuspended in 100 µL PBS 1× for cell detection with fluorescence microscopy, except for the experiment with 100 SK-BR-3 cells, where captured Ab/MNP-Si were detected directly with fluores-cence microscopy.
    3. Results
    3.1. Checking MNP characteristics
    In the first step the properties and characteristics of MNPs were verified with various tests to confirm effective coating with APTES.
    3.1.1. Vibrating sample magnetometer (VSM)
    To investigate the magnetic properties of nanoparticles after coating, VSM tests were carried out to record the steady magnetic field
    (H) which induces magnetic moment (m) in the sample and the hys-teresis loop before and after coating with APTES. As shown in Fig. 1, the presence of a very small hysteresis loop shows that the MNPs alone had paramagnetic properties very close to superparamagnetism, which re-sults in the rapid precipitation of water-dispersed MNPs in the presence of a low magnetic field. Based on the literature, the smaller squareness ratio or remanence to saturation magnetization (Mr/Ms) indicates greater magnetic properties, and materials with a squareness ratio equal to zero are considered as superparamagnetic [18,46]. The squareness ratio of our MNPs was about 0.1, which indicates their near to superparamagnetic properties. However, the level of saturated
    Fig. 8. Flow cytometry diagrams forSK-BR-3 cell isolation from different concentrations (0.1– 4 × 105) in a mixture of peripheral mononuclear cells. Right column: SK-BR-3 cells after magnetic separation; left column: control without separation.
    Table 1
    The efficiency of Ab/MNP-Si in isolating target cells from different concentra-tions of SK-BR-3 cells, evaluated by flow cytometry.
    Concentration of SK-BR-3 SK-BR-3 cells
    With washing (%) Without washing Yield (%)
    magnetism of the coated MNPs was lower compared to the pure MNPs by about 5 emu/g (64.5 emu/g vs. 69.5 emu/g), which indicates that the coating has very little effect on the magnetic properties of the particles. This decrease maybe explained by the nonmagnetic layer of APTES on the surface of MNP-Si. It also indicates that coating on the surface of the MNPs was effective.
    [18,36]. These peaks also indicate that despite the coating of MNPs with APTES, their crystalline structure had not undergone any changes. To further investigate the crystalline structure, the Standard Graph of iron oxide MNPs (JCPDS 19-0629 of magnetite) is given for comparison [47]. Average crystallite size was calculated with the Debye–Sherrer equation: D = k λ/β cosθ, which is illustrated by the peak at 2θ = 35.7 in Fig. 2 [47,48].The average size of MNPs was about 39.14 nm, which is within the range of sizes declared in the product datasheet for MNPs.
    D indicates the average crystallite size, k = 0.9 is Sherrer’s constant, λ is the wavelength, β indicates the full width at half-maximum of the highest intensity reflection, and θ is the Bragg diffraction angle.
    3.1.3. Fourier-transformed infrared spectroscopy (FT-IR)
    FT-IR was also conducted to further characterize silane bonding to MNPs (Fig. 3) by comparing the FT-IR spectrum of MNP and MNP-Si. The pure MNPs have a PVP coating, and according to the CeN and C]