Magnetic Nanoclusters Coated with Albumin, Casein, and Gelatin: Size Tuning, Relaxivity, Stability, Protein Corona, and Application in Nuclear Magnetic Resonance Immunoassay

Abstract

The surface functionalization of magnetic nanoparticles improves their physicochemical properties and applicability in biomedicine. Natural polymers, including proteins, are prospective coatings capable of increasing the stability, biocompatibility, and transverse relaxivity (r2) of magnetic nanoparticles. In this work, we functionalized the nanoclusters of carbon-coated iron nanoparticles with four proteins: bovine serum albumin, casein, and gelatins A and B, and we conducted a comprehensive comparative study of their properties essential to applications in biosensing. First, we examined the influence of environmental parameters on the size of prepared nanoclusters and synthesized protein-coated nanoclusters with a tunable size. Second, we showed that protein coating does not significantly influence the r2 relaxivity of clustered nanoparticles; however, the uniform distribution of individual nanoparticles inside the protein coating facilitates increased relaxivity. Third, we demonstrated the applicability of the obtained nanoclusters in biosensing by the development of a nuclear-magnetic-resonance-based immunoassay for the quantification of antibodies against tetanus toxoid. Fourth, the protein coronas of nanoclusters were studied using SDS-PAGE and Bradford protein assay. Finally, we compared the colloidal stability at various pH values and ionic strengths and in relevant complex media (i.e., blood serum, plasma, milk, juice, beer, and red wine), as well as the heat stability, resistance to proteolytic digestion, and shelf-life of protein-coated nanoclusters.

Keywords: antibody; assay; colloidal stability; nanoparticles; protein; protein G; streptavidin.

Figure 1

Synthesis of Fe@C-NH₂/Protein/Str and Fe@C-NH₂/Protein/G. 4-ABA-4-aminobenzylamine.

Figure 2

Relaxivity of conjugates with different sizes and coating types, n = 5, mean ± SD. Letters “S”, “M”, and “L” indicate “small”, “medium”, and “large” nanoclusters, respectively; the dashed line indicates the relaxivity of the parent Fe@C-NH₂ (285 1/mM⁻¹ × s⁻¹). Conjugate Fe@C-NH₂/Gelatin B/Str with highest relaxivity was excluded when mean relaxivity values were calculated.

Figure 3

TEM images of Fe@C-NH₂/Casein/Str (a), Fe@C-NH₂/BSA/Str (b), and Fe@C-NH₂/Gelatin B/Str (c). Scale bars are 20 nm.

Figure 4

Properties of nanoclusters coated with different proteins. Upper left: atomic force microscopy (AFM) images of nanoclusters coated with different proteins: (a) BSA, (b) casein, (c) gelatin A, and (d) gelatin B; lower left: (e) thermogravimetric analysis (TGA) curves of Fe@C-NH₂, BSA and Fe@C-NH₂/BSA/Str in airflow; upper right: UV-Vis spectra of Fe@C-NH₂, Fe@C-NH₂/Protein/Str and proteins: (f) BSA, (g) casein, (h) gelatin B, and (i) gelatin A; and lower right: (j) stability of Fe@C, Fe@C-NH₂, Fe@C-NH₂/Gelatin B/Str in buffers with pH 4 and 7.

Figure 5

Application of protein-coated magnetic nanoclusters in nuclear magnetic resonance (NMR)-immunoassay of IgG against the anti-tetanus toxoid. (a) the principle of the assay; (b) day-to-day variability of the anti-TT NMR-assay; (c) dose-response curve obtained using Fe@C-NH₂/Casein/G; (d) NMR-relaxometer and sample holder (inset): 1—magnet, 2—sample holder, 3—NMR-relaxometer, 4—radio-frequency coil, and 5—test-strip in plastic envelope.

Figure 6

Thermal stability of (BSA) Fe@C-NH₂/BSA/G; (Casein) Fe@C-NH₂/Casein/G; (Gel B) Fe@C-NH₂/Gelatin B/G; (Gel A) Fe@C-NH₂/Gelatin A/G. Solid line—hydrodynamic diameter; dashed line—polydispersity index, n = 3, mean ± SD.

Figure 7

Colloidal stability of Fe@C-NH₂/Protein/G in buffers with different pH and ionic strength values. Ionic strength values are specified at the top of the figure. “BSA”, “Casein”, “Gel B”, and “Gel A” indicate coating protein. Blue line—hydrodynamic diameter at 0 (dashed line) and 24 h (solid line); orange line—polydispersity index at 0 (dashed line) and 24 h (solid line), n = 3, mean ± SD.

Figure 8

The T2 relaxation times of Fe@C-NH₂/Protein/G diluted in buffers with different pH and ionic strength values and the zeta potentials of Fe@C-NH₂/Protein/G. “BSA”, “Casein”, “Gel B”, and “Gel A” indicate coating protein. T2 in 0.15 M (red line), 0.5 M (green line), and 2 M (blue line) buffers at 0 (dashed line) and 24 h (solid line), n = 3, mean ± SD.

Figure 9

Storage stability of Fe@C-NH₂/Protein/G and stability of Fe@C-NH₂/Protein/G and Fe@C-NH₂/Protein/Str in complex media. Upper row: changes in hydrodynamic diameter (a) and polydispersity index (b) during four weeks of storage at different temperatures; the five bars represent the size or PdI at week 0, 1, 2, 3, and 4 (from left to right), statistics: two-way ANOVA with Dunnet’s post-hoc test, n = 3, mean ± SD; lower row: the T2 of Fe@C-NH₂/Protein/G (c) and Fe@C-NH₂/Protein/Str (d) diluted in juice (J), wine (W), beer (B), milk (M), blood serum (S), and plasma (P); the three bars represent T2 at 0, 1, and 5 h (from left to right).

Figure 10

Protein corona and stability of nanoclusters to proteolysis. Protein coronas of Fe@C-NH₂/BSA/Str (lane 2), Fe@C-NH₂/Casein/Str (lane 3), Fe@C-NH₂/Gelatin A/Str (lane 4), Fe@C-NH₂/Gelatin B/Str (lane 5), and Fe@C-NH₂ (lane 6) in blood serum (a) and plasma (c), with lane 1-protein markers (kDa). (b) The sorption of serum proteins on Fe@C-NH₂/Protein/Str and Fe@C-NH₂. (d) The size of Fe@C-NH₂/Protein/Str after incubation in the trypsin solution (dashed line, filled circles) or PBS (solid line, open circles). Coating: BSA (red), casein (yellow), gelatin A (green) and B (blue), n = 3, mean ± SD.