br Thermogravimetric analysis TGA br To
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 , 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 . 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.
3.1.6. Scanning T-5224 microscopy (SEM)
The morphology and size distribution of MNPs were investigated by SEM analysis. SEM images showed a uniform pattern in both MNP-Si and MNPs, with a similar size distribution (Fig. 6).
3.2. Herceptin conjugation to MNP-Si
As shown in scheme 1A, to coat the surface of MNP-Si with
Fig. 10. Fluorescence microscopy images of 100 stained SK-BR-3 cells in whole blood after magnetic separation. a,c) Without, and b,d) with fluorescent lighting. c,d) With local magnification.
Herceptin, antibodies need to be initially activated with EDC/NHS be-cause MNP-Si has an amine group (reverse direction). The MNP-Si were added to the active antibodies, and an unstable intermediate product was formed which reacted with the primary amine group, and then an amide bond was formed between the two materials. Studies have shown that antibodies have two types of amines: primary amines will settle at the site of antibody binding to its antigen, and a large number of sec-ondary amines will be located in other sites. EDC/NHS-activated acid reacts with primary amines . Consequently, if the MNPs contain an acidic group and are initially activated by EDC/NHS, they will react with the primary amine in a specific site (paratope), thereby reducing the antibody’s ability to conjugate to the antigen. In the reverse di-rection this problem was resolved, because the specific areas or para-topes did not react. The MNPs in this study did not contain an acidic group, so the reverse reaction approach was used, with interesting re-sults as explained below.
Since Herceptin is a human IgG , an anti-human IgG1 antibody conjugated to a fluorescent dye was used to verify the conjugation of Herceptin to MNPs, and changes in fluorescence intensity observed for MNP-Si with and without Herceptin were considered as a criterion for eﬃcient coating. In many previous studies, complicated and time-consuming tests such as gel electrophoresis, the Bradford test, western blotting and SDS-PAGE have been used to investigate the binding of a protein or antibody to a nanoparticle [7,59]. But here we applied a flow cytometric-based method which is not only an accurate and reliable method, but also an easier and faster one compared to other ap-proaches.