*3.2. Synthesis of Fe3O<sup>4</sup> NPs*

The NPs were synthesized using the coprecipitation method [37]. A mixture of acidic solutions (HCl, 2 M) of the FeCl2·2H2O (5 mL, 5 M) and FeCl3·6H2O (20 mL, 2.5 M) salts was added to an aqueous solution of NH<sup>3</sup> (250 mL, 0.7 M) contained in a three-neck balloon using a peristaltic pump. All the solutions were bubbled with N<sup>2</sup> (g) before the synthesis. The reaction mixture was kept at room temperature under an N<sup>2</sup> (g) atmosphere under mechanical stirring (500 rpm) and ultrasound-treated for 25 min. After the reaction time, the obtained MNPs were separated using a neodymium magnet. Subsequently, they were washed with 100 mL of deionized water and separated again to obtain a black powder corresponding to Fe3O4, which was dried in a desiccator containing activated silica for 12 h.

## *3.3. Coating/Functionalization of Fe3O<sup>4</sup> NPs*

## 3.3.1. Fe3O4@citrate

The functionalization of the MNPs as carboxylic acids was carried out by coating Fe3O<sup>4</sup> with sodium citrate. Fe3O<sup>4</sup> (500 mg) was added to an aqueous solution of Na3C6H5O7·2H2O (50 mL, 1 mM). The reaction mixture was mechanically stirred for 4 h at room temperature, and then adjusted to pH = 9 using an aqueous solution of NH<sup>3</sup> (0.7 M). The obtained precipitate was magnetically separated and washed twice with 50 mL of deionized water. Finally, it was dried (as explained for the synthesis of the Fe3O<sup>4</sup> NPs), and Fe3O4@citrate was obtained in the form of a black powder.

## 3.3.2. Fe3O4@APTES

Additionally, the functionalization of the MNPs as amines was carried out by modifying the surface of Fe3O<sup>4</sup> with APTES [31]. Briefly, 500 mg of Fe3O<sup>4</sup> were redispersed in a mixture of ethanol (80 mL) and deionized water (40 mL). The dispersion was adjusted to pH = 10 with an aqueous solution of NH<sup>3</sup> (0.7 M) and homogenized in an ultrasonic bath. Subsequently, an alcoholic solution of APTES (5 mL, 10%) was dripped to the reaction mixture under constant stirring. After 24 h, the precipitate was separated with a neodymium magnet and washed twice with 50 mL of ethanol. Finally, the product was dried according to the procedure described above to obtain Fe3O4@APTES in the form of a black powder.

### *3.4. Cu(II) Adsorption Capacity of Functionalized NPs*

Once the functionalization of MNPs was completed, the degree of substitution on the magnetic oxide was calculated. The determination could be carried out using the bicinchoninate method from the quantification of Cu(II) ions remaining in aqueous dispersions [38]. An aqueous solution of CuSO4·5H2O (35.5 mL, 100 mg/L) was prepared in an Erlenmeyer flask. Subsequently, 5 mL of the solution were collected, and a bicinchoninic acid kit was added. The absorbance was measured using an ultraviolet (UV)–visible (Vis) spectrophotometer at 560 nm (time: 0 h), and then the concentration was determined. The functionalized MNP sample (0.03 g) was added to the remaining solution (30.5 mL) and the pH was adjusted to 5 with HCl (0.1 M). The reaction mixture was mechanically stirred with a glass-lined magnetic stirrer at 140 rpm for 1 h. The magnetic system was then separated with a neodymium magnet. The supernatant (0.25 mL) was then collected, filtered, and diluted with 4.75 mL of distilled water, and a bicinchoninic acid kit was added. The absorbance was measured at 560 nm (time: 1 h), and then the concentration was determined. The last absorbance measurement was performed 24 h after the functionalized MNPs were in contact with CuSO4·5H2O (time: 24 h). Each measurement was performed in duplicate.

## *3.5. Synthesis of Peptide–Fe3O<sup>4</sup> Conjugates by the U-4C Reaction*

To conjugate the peptide fractions to Fe3O<sup>4</sup> using the U-4C reaction, the amino component (the peptide fractions or Fe3O4@APTES) and four equivalents (eq) of paraformaldehyde were reacted at room temperature for 24 h in the smallest required volume of phosphate-buffered saline (PBS) (pH = 7.4). The acid component (Fe3O4@citrate or peptide fractions) was then added; 10 min later, 1-pentynylisonitrile (4 eq) was added. The reactions were monitored at intervals of 4 h over 8 h and carried out in quintuplicate. Finally, the products were washed twice with 1 mL of distilled water and dried, as stated for the synthesis of Fe3O<sup>4</sup> NPs.

### *3.6. Extension of the Conjugation*

The amount of peptide remaining after conjugation to MNPs by the U-4C reaction was determined according to the Bradford assay [39]. The supernatants of each reaction (10 µL) were deposited with 100 µL of the Bradford reagent in a 96-well microplate. The readings were carried out at a wavelength of 595 nm. The remaining peptide concentration values were determined using a calibration curve of bovine serum albumin (BSA).

### *3.7. NP Characterizations Using X-ray Diffraction, Fourier-Transform Infrared Spectroscopy, Scanning Transmission Electron Microscopy, Thermogravimetric Analysis, Dynamic Light Scattering, and Spectrophotometric Analysis*

XRD measurements were performed using a Bruker D8 Advance diffractometer. Cu *Kα* (*λ* = 1.54183 Å) incident radiation was used. The measurements were carried out in the range of 5–100◦ with an increment of 0.02◦ and scan speed of 10 s. The cell parameters were calculated using the Powdercell 2.4 software. The average crystallite size was determined using the Debye–Scherrer equation (Equation (1))

$$D = \frac{\kappa \lambda}{\beta \cos \theta}.\tag{1}$$

where *D* is the crystallite size, *κ* is the Debye–Scherrer constant (0.89), *λ* is the operation wavelength, *β* is the average width of the most intense peak, and *θ* is the Bragg angle.

FTIR spectra were recorded using an Equinox 55 Bruker spectrometer in the absorbance measurement range of 4000 to 400 cm−<sup>1</sup> . TGA measurements were performed using a Netzsch STA 409 PC Luxx thermal analyzer coupled to the FTIR equipment. Approximately 10 mg of the sample were heated from 25 to 1000 ◦C and analyzed at the heating flux of 20 ◦C/min in an Ar atmosphere. DLS measurements were carried out using a Zetasizer Nano sampler (Malvern Instruments, Cambridge, UK) equipped with a He–Ne laser (λ = 633 nm). The measurements were carried out in square polystyrene cells at 25 ◦C in the range of 0.3–10 µm using H2O as a dispersion medium. The absorbance performance was assessed using two different types of equipment. To determine the adsorption capacity of Cu2+ ions, the absorbance values were obtained at 560 nm using a Genesys 10 UV spectrophotometer (Thermo Electron Corporation, Madison, WI, USA). During the experiment with the peptide fractions, the absorbances were measured using a microplate reader (Thermo Scientific Multiskan GO, Waltham, MA, USA). The readings were carried out at 536, 595, and 700 nm depending on the system and properties to be determined. The morphology and average size of the NPs were analyzed using STEM images. The measurements were carried out using a JEM-ARM200CF electronic microscope with the resolution of 80 pm for the employed mode. The acceleration voltage was 200 keV. The analysis of the STEM images to determine the NP average size was carried out with the ImageJ (Origin(Pro) Corporation, Northampton, MS, USA), and the histograms were obtained using Origin v9.0, (OriginLab Corporation, Northampton, MS, USA).
