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

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.


Experimental section
Synthesis of "small" (100-110 nm) BSA-coated Fe@C-NH2 nanoparticles conjugated with streptavidin 325 μL of 20% BSA in H2O was mixed with 975 μL of the 0.1M acetic buffer with pH 4, then 1300 μL of 10 mg/ml Fe@C-NH2 suspension was added dropwise under vortex stirring. pH was adjusted to 7.25 with 1M NaOH. Resulting suspension (2500 μL) was added dropwise under vortex stirring to an equal volume of 25% glutaraldehyde solution (see Materials section) and incubated on rotating mixer (from now on rotation angle was set at 99 degrees, the rotation rate was 5 rpm) at room temperature for 30 min. Then, 5 ml of nanoparticles activated with glutaraldehyde were passed through the chromatography column (column XK 26/40, medium: Sepharose CL-6B, bed volume: 160 ml, elution speed: 0.65 ml/min, eluent: PBS) to remove the excess of BSA and glutaraldehyde. Fractions containing Fe@C-NH2/BSA nanoparticles were collected and concentrated to approximately 2.5 ml. For concentrations, the suspension was placed in dialysis tubing (10 kDa MWCO, 2 ml per cm) and covered with the layer of 35 kDa PEG. In 2-3 hours concentrated suspension was removed from dialysis tubing and centrifuged at 1600g for 5 min. The supernatant contained Fe@C-NH2/BSA nanoparticles activated with glutaraldehyde was divided into five equal portions (volume of the portion was approximately 500 μL). Each portion was added to 10 mg/ml streptavidin solution in PBS under vortex stirring; volumes were equalized with PBS. Final streptavidin to Fe@C-NH2/BSA ratios were 10, 20, 40, 80 and 160 μg of streptavidin per 1 mg of nanoparticles. Conjugation of streptavidin was carried out overnight on rotating mixer at +4 o C. Unreacted streptavidin was removed by gel-chromatography (column C 10/20, medium: Sepharose CL-6B, bed volume: 10 ml, elution speed: 0.07 ml/min, eluent: PBS). Fractions with the highest concentration of Fe@C-NH2/BSA/Str nanoparticles were combined. 325 μL of 20% BSA in H2O was mixed with 975 μL of 0.1M phosphate buffer pH 8, then 1300 μL of 10 mg/ml Fe@C-NH2 suspension was added dropwise under vortex stirring. pH was adjusted to 7.25 with 1M HCl. Resulting suspension (2500 μL) was added dropwise under vortex stirring to an equal volume of 25% glutaraldehyde solution (see Materials section) and incubated on rotating mixer at room temperature for 30 min. Further procedures were as described for "small" Fe@C-NH2/BSA/Str (see above).
Synthesis of "large" (210-220 nm) BSA-coated Fe@C-NH2 nanoparticles conjugated with streptavidin 325 μL of 20% BSA in H2O was mixed with 975 μL of 0.1M phosphate buffer pH 8, then ionic strength of mixture was adjusted to 0.5M with NaCl powder. After that, 1300 μL of 10 mg/ml Fe@C-NH2 suspension was added dropwise under vortex stirring. pH was adjusted to 7.25 with 1M HCl. Resulting suspension (2500 μL) was added dropwise under vortex stirring to an equal volume of 25% glutaraldehyde solution (see Materials section) and incubated on rotating mixer at room temperature for 30 min. Further procedures were as described for "small" Fe@C-NH2/BSA/Str (see above).
Synthesis of "small" (110-120 nm) casein-coated Fe@C-NH2 nanoparticles conjugated with streptavidin 738 μL of 8.8% casein in H2O was mixed with 562 μL of 0.1M acetic buffer pH 5, then 1300 μL of 10 mg/ml Fe@C-NH2 suspension was added dropwise under vortex stirring. pH was adjusted to 7.25 with 1M HCl. Resulting suspension (2500 μL) was sonicated for 10 sec (30% amplification, 3 mm probe), added dropwise under vortex stirring to an equal volume of 25% glutaraldehyde solution (see Materials section) and incubated on rotating mixer at room temperature for 30 min. Further procedures were as described for "small" Fe@C-NH2/BSA/Str (see above).
Synthesis of "medium" (190-210 nm) casein-coated Fe@C-NH2 nanoparticles conjugated with streptavidin 738 μL of 8.8% casein in H2O was mixed with 562 μL of 0.1M acetic buffer pH 5, then ionic strength of mixture was adjusted to 0.5M with NaCl powder. After that, 1300 μL of 10 mg/ml Fe@C-NH2 suspension was added dropwise under vortex stirring. pH was adjusted to 7.25 with 1M HCl. Resulting suspension (2500 μL) was added dropwise under vortex stirring to an equal volume of 25% glutaraldehyde solution (see Materials section) and incubated on rotating mixer at room temperature for 30 min. Further procedures were as described for "small" Fe@C-NH2/BSA/Str (see above).

Synthesis of "large" (240-260 nm) casein-coated Fe@C-NH2 nanoparticles conjugated with streptavidin
738 μL of 8.8% casein in H2O was mixed with 562 μL of 0.1M acetic buffer pH 5, then ionic strength of mixture was adjusted to 1M with NaCl powder. After that, 1300 μL of 10 mg/ml Fe@C-NH2 suspension was added dropwise under vortex stirring. pH was adjusted to 7.25 with 1M HCl. Resulting suspension (2500 μL) was sonicated for 10 sec (30% amplification, 3 mm probe), added dropwise under vortex stirring to an equal volume of 25% glutaraldehyde solution (see Materials section) and incubated on rotating mixer at room temperature for 30 min. Further procedures were as described for "small" Fe@C-NH2/BSA/Str (see above).

Synthesis of "small" (140-160 nm) gelatin B-coated Fe@C-NH2 nanoparticles conjugated with streptavidin
650 μL of 10% gelatin B in H2O was mixed with 650 μL of 0.1M acetic buffer pH 4, then 1300 μL of 10 mg/ml Fe@C-NH2 suspension was added dropwise under vortex stirring. pH was adjusted to 7.25 with 1M NaOH. Resulting suspension (2500 μL) was sonicated on a water bath for 5 min (30% amplification, 3 mm probe), added dropwise under vortex stirring to an equal volume of 25% glutaraldehyde solution (see Materials section) and incubated on rotating mixer at +37 o C for 30 min. All solutions were pre-warmed and kept at +37-40 o C to prevent gelation. Further procedures were performed at room temperature as described for "small" Fe@C-NH2/BSA/Str (see above).

Synthesis of "medium" (210-240 nm) gelatin B-coated Fe@C-NH2 nanoparticles conjugated with streptavidin
650 μL of 10% gelatin B in H2O was mixed with 650 μL of 0.1M phosphate buffer pH 8, then ionic strength of mixture was adjusted to 1M with NaCl powder. After that, 1300 μL of 10 mg/ml Fe@C-NH2 suspension was added dropwise under vortex stirring. Resulting suspension (2500 μL) was sonicated on a water bath for 2 sec (30% amplification, 3 mm probe), added dropwise under vortex stirring to an equal volume of 25% glutaraldehyde solution (see Materials section) and incubated on rotating mixer at +37 o C for 30 min. All solutions were pre-warmed and kept at +37-40 o C to prevent gelation. Further procedures were performed at room temperature as described for "small" Fe@C-NH2/BSA/Str (see above).
Synthesis of "large" (300-320 nm) gelatin B-coated Fe@C-NH2 nanoparticles conjugated with streptavidin 1300 μL of 10 mg/ml Fe@C-NH2 suspension was mixed with 650 μL of 0.1M phosphate buffer pH 8, then 650 μL of 10% gelatin B in H2O was added dropwise under vortex stirring. Resulting suspension (2500 μL) was sonicated on water bath for 5 sec (30% amplification, 3 mm probe), centrifuged at 1600g for 5 min to remove large agglomerates, then supernatant was sonicated for another 5 sec and added dropwise under vortex stirring to equal volume of 25% glutaraldehyde solution (see Materials section) and incubated on rotating mixer at +37 o C for 30 min. All solutions were pre-warmed and kept at +37-40 o C to prevent gelation. Further procedures were performed at room temperature as described for "small" Fe@C-NH2/BSA/Str (see above).

Synthesis of BSA-coated Fe@C-NH2 functionalized with Streptococcal protein G (Fe@C-NH2/BSA/G)
2250 μL of 10 mg/ml Fe@C-NH2 suspension was added dropwise under vortex stirring to 625 μL of 20% BSA in H2O, then 1595 μL of PBS and 30 μL of 1M NaOH were added to adjust pH to 7.2-7.6. Resulting suspension (4300 μL) was sonicated for 10 s and added dropwise under vortex stirring to an equal volume of 25% glutaraldehyde solution and incubated on rotating mixer at room temperature for 30 min. Then, 8.6 ml of nanoparticles activated with glutaraldehyde were passed through the chromatography column (column XK 26/40, medium: Sepharose CL-6B, bed volume: 160 ml, elution speed: 0.65 ml/min, eluent: PBS) to remove the excess of BSA and glutaraldehyde. Fractions containing Fe@C-NH2/BSA nanoparticles were collected and concentrated to approximately 3-4 ml. For concentrations, the suspension was placed in dialysis tubing (10 kDa MWCO, 2 ml per cm) and covered with the layer of 35 kDa PEG. After 2-3 hours concentrated suspension was removed from dialysis tubing and centrifuged at 1600g for 5 min. The supernatant contained Fe@C-NH2/BSA nanoparticles activated with glutaraldehyde was added to 10 mg/ml protein G solution in PBS under vortex stirring. Final protein G to Fe@C-NH2/BSA ratio was 80 μg of protein G per 1 mg of nanoparticles. Conjugation of protein G was carried out overnight on rotating mixer at +4 o C, glycine was added to 6 mM to quench unreacted aldehyde groups and the mixture was incubated at RT for one more hour. Unreacted protein G was removed by gel-chromatography (column XK 26/40, medium: Sepharose CL-6B, bed volume: 100 ml, elution speed: 0.65 ml/min, eluent: PBS). Fractions with the highest concentration of Fe@C-NH2/BSA/Str nanoparticles were combined. Glycerol, BSA and glycine were added to the final concentrations of 20%, 1%, and 6 mM respectively. Conjugates were stored at +4 o C. The concentration of nanoparticles in prepared conjugates were determined by absorbance at 450 nm.

Synthesis of casein-coated Fe@C-NH2 functionalized with Streptococcal protein G (Fe@C-NH2/Casein/G)
1278 μL of 8.8% casein in H2O was mixed with 962 μL 0.1M acetic buffer pH 5 and 9.6 μL of 1M NaOH. After that, 2250 μL of 10 mg/ml Fe@C-NH2 suspension was added dropwise under vortex stirring. Resulting suspension (4300 μL) was sonicated for 10 s and added dropwise under vortex stirring to an equal volume of 25% glutaraldehyde solution and incubated on rotating mixer at room temperature for 30 min. Then, 8.6 ml of nanoparticles activated with glutaraldehyde were passed through the chromatography column (column XK 26/40, medium: Sepharose CL-6B, bed volume: 160 ml, elution speed: 0.65 ml/min, eluent: PBS) to remove the excess of casein and glutaraldehyde. Fractions containing Fe@C-NH2/Casein nanoparticles were collected and concentrated to approximately 3-4 ml. For concentration, the suspension was placed in dialysis tubing (10 kDa MWCO, 2 ml per cm) and covered with the layer of 35 kDa PEG. After 2-3 hours concentrated suspension was removed from dialysis tubing and centrifuged at 1600g for 5 min. The supernatant contained Fe@C-NH2/Casein nanoparticles activated with glutaraldehyde was added to 10 mg/ml protein G solution in PBS under vortex stirring. Final protein G to Fe@C-NH2/Casein ratio was 80 μg of protein G per 1 mg of nanoparticles. Conjugation of protein G was carried out overnight on rotating mixer at +4 o C, glycine was added to 6 mM to quench unreacted aldehyde groups, and the mixture was incubated at RT for one more hour. Unreacted protein G was removed by gel-chromatography (column XK 26/40, medium: Sepharose CL-6B, bed volume: 100 ml, elution speed: 0.65 ml/min, eluent: PBS). Fractions with the highest concentration of Fe@C-NH2/BSA/Str nanoparticles were combined. Glycerol, BSA and glycine were added to the final concentrations of 20%, 1%, and 6 mM respectively. Conjugates were stored at +4 o C. The concentration of nanoparticles in prepared conjugates was determined by absorbance at 450 nm.

Synthesis of Gelatin A/B-coated Fe@C-NH2 functionalized with Streptococcal protein G (Fe@C-NH2/Gelatin A/G and Fe@C-NH2/Gelatin B/G)
1125 μL of 10% gelatin in H2O was mixed with 1125 μL 0.1M acetic buffer pH 4. After that, 2250 μL of 10 mg/ml Fe@C-NH2 suspension was added dropwise under vortex stirring; pH was adjusted to 7.25 by 1M NaOH. Resulting suspension (4300 μL) was sonicated for 5 min and added dropwise under vortex stirring to an equal volume of 25% glutaraldehyde solution and incubated on rotating mixer at +37 o C for 30 min. All solutions were pre-warmed and kept at +37-40 o C to prevent gelation. Then, 8.6 ml of nanoparticles activated with glutaraldehyde were passed through the chromatography column (column XK 26/40, medium: Sepharose CL-6B, bed volume: 160 ml, elution speed: 0.65 ml/min, eluent: PBS) to remove the excess of gelatin and glutaraldehyde. Fractions containing Fe@C-NH2/Gelatin B nanoparticles were collected and concentrated to approximately 3-4 ml. For concentration, the suspension was placed in dialysis tubing (10 kDa MWCO, 2 ml per cm) and covered with the layer of 35 kDa PEG. After 2-3 hours concentrated suspension was removed from dialysis tubing and centrifuged at 1600g for 5 min. The supernatant contained Fe@C-NH2/Gelatin B nanoparticles activated with glutaraldehyde was added to 10 mg/ml protein G solution in PBS under vortex stirring. Final protein G to Fe@C-NH2/Gelatin B ratio was 80 μg of protein G per 1 mg of nanoparticles. Conjugation of protein G was carried out overnight on rotating mixer at +4 o C, glycine was added to 6 mM to quench unreacted aldehyde groups, and the mixture was incubated at RT for one more hour. Unreacted protein G was removed by gel-chromatography (column XK 26/40, medium: Sepharose CL-6B, bed volume: 100 ml, elution speed: 0.65 ml/min, eluent: PBS). Fractions with the highest concentration of Fe@C-NH2/BSA/Str nanoparticles were combined. Glycerol, BSA and glycine were added to the final concentrations of 20%, 1%, and 6 mM respectively. Conjugates were stored at +4 o C. The concentration of nanoparticles in prepared conjugates was determined by absorbance at 450 nm.

Synthesis of BSA-coated Fe@C-NH2 functionalized with streptavidin (Fe@C-NH2/BSA/Str)
2250 μL of 10 mg/ml Fe@C-NH2 suspension was added dropwise under vortex stirring to 625 μL of 20% BSA in H2O, then 1595 μL of PBS and 30 μL of 1M NaOH were added to adjust pH to 7.2-7.6. Resulting suspension (4300 μL) was sonicated for 10 s and added dropwise under vortex stirring to an equal volume of 25% glutaraldehyde solution and incubated on rotating mixer at room temperature for 30 min. Then, 8.6 ml of nanoparticles activated with glutaraldehyde were passed through the chromatography column (column XK 26/40, medium: Sepharose CL-6B, bed volume: 160 ml, elution speed: 0.65 ml/min, eluent: PBS) to remove the excess of BSA and glutaraldehyde. Fractions containing Fe@C-NH2/BSA nanoparticles were collected and concentrated to approximately 3-4 ml. For concentrations, the suspension was placed in dialysis tubing (10 kDa MWCO, 2 ml per cm) and covered with the layer of 35 kDa PEG. After 2-3 hours concentrated suspension was removed from dialysis tubing and centrifuged at 1600g for 5 min. The supernatant contained Fe@C-NH2/BSA nanoparticles activated with glutaraldehyde was added to 10 mg/ml streptavidin solution in PBS under vortex stirring. Final streptavidin to Fe@C-NH2/BSA ratio was 80 μg of streptavidin per 1 mg of nanoparticles. Conjugation of streptavidin was carried out overnight on rotating mixer at +4 o C, glycine and BSA were added to 6 mM and 1 mg/ml respectively to quench unreacted aldehyde groups, and the mixture was incubated at RT for one more hour. Unreacted streptavidin was removed by gel-chromatography (column XK 26/40, medium: Sepharose CL-6B, bed volume: 100 ml, elution speed: 0.65 ml/min, eluent: PBS). Fractions with the highest concentration of Fe@C-NH2/BSA/Str nanoparticles were combined. Conjugates were stored at +4 o C in PBS without any stabilizers. The concentration of nanoparticles in prepared conjugates were determined by absorbance at 450 nm.

Synthesis of casein-coated Fe@C-NH2 functionalized with streptavidin (Fe@C-NH2/Casein/Str)
1278 μL of 8.8% casein in H2O was mixed with 962 μL 0.1M acetic buffer pH 5 and 9.6 μL of 1M NaOH. After that, 2250 μL of 10 mg/ml Fe@C-NH2 suspension was added dropwise under vortex stirring. Resulting suspension (4300 μL) was sonicated for 10 s and added dropwise under vortex stirring to an equal volume of 25% glutaraldehyde solution and incubated on rotating mixer at room temperature for 30 min. Then, 8.6 ml of nanoparticles activated with glutaraldehyde were passed through the chromatography column (column XK 26/40, medium: Sepharose CL-6B, bed volume: 160 ml, elution speed: 0.65 ml/min, eluent: PBS) to remove the excess of casein and glutaraldehyde. Fractions containing Fe@C-NH2/Casein nanoparticles were collected and concentrated to approximately 3-4 ml. For concentrations, the suspension was placed in dialysis tubing (10 kDa MWCO, 2 ml per cm) and covered with the layer of 35 kDa PEG. After 2-3 hours concentrated suspension was removed from dialysis tubing and centrifuged at 1600g for 5 min. The supernatant contained Fe@C-NH2/Casein nanoparticles activated with glutaraldehyde was added to 10 mg/ml streptavidin solution in PBS under vortex stirring. Final streptavidin to Fe@C-NH2/Casein ratio was 80 μg of streptavidin per 1 mg of nanoparticles. Conjugation of streptavidin was carried out overnight on rotating mixer at +4 o C, glycine was added to 6 mM to quench unreacted aldehyde groups, and the mixture was incubated at RT for one more hour. Unreacted streptavidin was removed by gelchromatography (column XK 26/40, medium: Sepharose CL-6B, bed volume: 100 ml, elution speed: 0.65 ml/min, eluent: PBS). Fractions with the highest concentration of Fe@C-NH2/Casein/Str nanoparticles were combined. Conjugates were stored at +4 o C in PBS without any stabilizers. The concentration of nanoparticles in prepared conjugates was determined by absorbance at 450 nm.