2.2.2. Characterization of Fe3O4@APTES

The functionalization of the Fe3O<sup>4</sup> NPs surface in the amine form was carried out from a silylation reaction with the use of APTES. This molecule is the most widely used alkoxysilane for coating magnetite NPs since it provides the highest density of amine groups on the surface of the magnetic core [31].

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The IR spectrum of Fe3O4@APTES showed characteristic bands that confirmed the presence of the expected APTES coating (Figure 3, green spectrum). Thus, a wide band, centered at 3399 cm−<sup>1</sup> , was observed, attributed to the overlapping valence vibrations of the O–H and N–H bonds of the hydroxyl and amine groups in APTES. The bands corresponding to the antisymmetric and symmetric vibrations of the APTES methylene groups appeared at 2928 and 2842 cm−<sup>1</sup> , respectively. The bands corresponding to the vibrations antisymmetric and symmetric of the NH<sup>2</sup> group in APTES were observed around 1642 and 1549 cm−<sup>1</sup> , respectively. The presence of silanol groups (Si–OH) and siloxanes (Si–O–Si) on the surface of the MNPs was verified by the bands of the valence vibrations of the Si–O–Si and Si–OH bonds at 1396 and 1015 cm−<sup>1</sup> , respectively. Finally, the Fe3O<sup>4</sup> band was maintained at 573 cm−<sup>1</sup> , suggesting the presence of magnetite in the conjugate. Moreover, no differences were observed between the XRD patterns of the coated and uncoated magnetite samples (Figure 1, green and black profile, respectively), which confirms that the crystallinity of the material was not affected by the coating with APTES.

The calculated cell parameter of Fe3O4@APTES was 8.3744 Å, slightly lower than that determined for the uncoated magnetite, as the Fe and O atoms are closer to each other in the unit cell owing to the APTES molecules [30]. The crystallite size of the APTES-coated NPs was 17 nm, which confirms that the compound maintained its nanoscale dimensions. This value is very similar to the particle size obtained using STEM (16 ± 3 nm), which

suggests that the system was monocrystalline. The STEM images (Figure 4b) show that the coated NPs have spherical morphology and are agglomerated because of their small sizes. The weight percentage of the APTES-coated Fe3O<sup>4</sup> was determined using TGA. A weight loss of 5.22% with respect to the uncoated Fe3O<sup>4</sup> was obtained, which confirmed the coating on the magnetite surface.

### *2.3. Determination of the Degree of Substitution of Functionalized MNPs*

The degree of substitution on the magnetic oxide was assessed, i.e., the amount of free functional groups on the surface of Fe3O4. For this purpose, the adsorption capacity of Cu (II) on the functionalized MNPs was determined using a spectrophotometric analysis. The results are presented in Table 1. To assess the molar quantity of free carboxylate groups on the MNPs coated with sodium citrate, it was considered that 2 M of carboxylate is necessary to complex 1 M of Cu2+. In the case of MNPs coated with APTES, it was assumed that the Cu2+ ions would form a complex with tetrahedral geometry with free amine groups on the magnetite [32]. Using this procedure, approximate molar quantities of free carboxylates or amine groups per gram of Fe3O<sup>4</sup> were obtained.

**Table 1.** Determination of the degree of substitution on the MNPs using the bicinchoninate method.


The calculations for the subsequent synthesis step were carried out using the average value of the loading of functional groups on the Fe3O<sup>4</sup> surface.
