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  • br In the first step the


    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 Geneticin, G-418 Sulfate 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]
    3.1.4. Thermogravimetric analysis (TGA)
    To verify silane binding to MNPs, TGA was carried out on MNP and MNP-Si. As seen in Fig. 4, in the sample of MNPs alone, weight loss occurred below 200 °C, between 200 °C and 500 °C, and above 550 °C, indicating the evaporation of water that was physically adsorbed, de-gradation of the PVP coating, and in third step, oxidation of magnetite (Fe3O4) to maghemite (Fe2O3) or the separation of hydroxyl ions from MNPs, respectively [51,53,54]. In MNP-Si samples, two steps of de-gradation could be observed. The greatest weight loss (1.7%) was ob-served at temperatures between 250 °C and 550 °C, and reflects the separation of amino-propyl groups in APTES from the MNP surface [51,52]. Another observed weight loss was similar to that in MNPs above 550 °C. Based on the literature [35], because the silane coated layer is thermally stable, all weight decreases in MNP-Si can be at-tributed essentially to iron oxide during the conversion of iron oxy-hydroxide to Fe2O3. Also, based on the literature, the weight loss due to PVP was observed at temperatures between 200 °C and 400 °C [48]. Therefore, the weight loss of PVP overlaps the main weight loss of MNP-Si in the TGA curve for MNP-Si [49,55]. The difference in weight loss in MNP was smaller than in MNP-Si in this range of temperatures (200–500 °C), which confirms the effective coating of MNPs by silane.
    To confirm silane coating on MNPs, EDX was used to analyze the elemental constituents of the MNPs before and after coating (Fig. 5). The presence of iron and oxygen was observed in two samples related to Fe3O4 (O peak at 0.53 and Fe peak at 6.41 keV), with iron more abundant than oxygen [23,24,56]. The presence of APTES on the sur-face of MNPs was confirmed by the increase in the percentages of C, Si and N: on uncoated MNPs the percentage of C was 0.27 (W%) and the values for both Si and N were zero, but on MNP-Si these values in-creased to 6.33, 0.67 and 4.44 (W%) respectively.