2.1. Synthesis of FCSNP and Characterization
A synthesis scheme for FCSNP is illustrated in Figure 1
. Functionalization with randomly distributed chemical groups for the addition of molecular bait was achieved by nucleophilic substitution (core) and the addition of an exclusion polymer (shell) by free radical polymerization. Each stage of synthesis was characterized by estimating the particle size and surface charge by dynamic light scattering (DLS) and zeta potential, respectively (Table 1
The average size of silica nanoparticles (Stage 1) was within the expected size range (50–900 nm) reported by Stöber [16
]. An increase in the nanoparticle size was obtained after the addition of APTES and the monomer 3-(Trimethoxysilyl) propyl methacrylate (stage 2). These reagents provide amino and ester functional groups, respectively, on the silica nanoparticles surface (core), which are necessary for the chemical modifications that followed. The organic compounds attached to the surface of the nanoparticles form a dense layer that increases the electrostatic repulsion at the surface, increasing the particle size [18
]. In this matter, Ferreira et al., 2015 reported an increase of ~70 nm after the modification of silica nanoparticles with APTES. This coincides with our results, since we obtained an increase of ~80 nm after the addition of both APTES and the monomer.
On the other hand, no change in the average size was obtained when Cibacron Blue was added (stage 3). This could be attributed to the hydrophobic nature of the molecule [12
]. Finally, when the exclusion polymer shell was formed (stage 4), the nanoparticle size increased due to the growth of the polymer chains [20
], thus forming the molecular sieve on the nanoparticle surface. In this grafting-from approach, crosslinked chains can be grown directly onto the particles to form the shell [21
], simply by the addition of a bi- or multifunctional crosslinker as a comonomer in the polymerization. The changes in the surface charge (zeta potential) after every functionalization stage (Table 1
) confirm the effective addition of each component [22
Further characterization included an Attenuated Total Reflection-Fourier Transformed Infrared (ATR-FTIR) analysis. Figure 2
shows the ATR-FTIR spectra of the silica nanoparticles and its consecutive modifications. In Figure 2
A, four significant peaks for the silica nanoparticles are shown. One peak at 1048 cm−1
is due to the asymmetric vibration of Si-O-Si stretching bands, and another peak at 794 cm−1
corresponds to Si-O-Si symmetric vibration [23
]. The next peak at 937 cm−1
corresponds to Si-OH and the last absorption peak around 3300 cm−1
is due to free O-H stretching bands [23
]. Some of these absorption peaks were also observed in the following modifications. In Figure 2
B, silica nanoparticles + APTES + monomer, new peaks appeared compared to the silica nanoparticle spectra. The 1630 cm−1
and 1457 cm−1
] related peaks are assigned to the NH2
deformation modes of the amine groups, while the 1718 cm−1
absorption peak corresponds to the C=O of the monomer [26
C shows the IR spectra of the nanoparticles after the addition of Cibacron blue. The absorption peaks at 1622 cm−1
and 1660 cm−1
are attributed to the vibration of N-H scissors [26
] and the peak at 1383 cm−1
is due to the C=N stretching vibration in CB [26
]. Finally, in Figure 2
D, after the addition of bis-acrylamide, two new absorption peaks at 1645 cm−1
and 1534 cm−1
appeared. The first peak corresponds to Amide I and represents the -C=O stretching vibration of the amide group [27
]. The second peak corresponds to Amide II, which is due to the -NH2
bending vibrations of the amine group [27
]. These signals indicate the successful modification of the nanoparticles at every stage of synthesis.
The morphological characterization of the first stage of synthesis performed by SEM (Figure 3
), showed a homogeneous distribution and the spherical shape of nanoparticles with a size of ~140 nm. This result was consistent with the size determined by dynamic light scattering (138.9 ± 5.6 nm).
The last stage of synthesis of FCSNP was characterized by TEM. Spherical nanoparticles of ~250 nm with a polymeric shell formed at the surface were observed (Figure 4
). Size was consistent with dynamic light scattering (243.9 ± 11.6 nm) data. The polymer layer thickness with an average of 38 nm was estimated from Figure 4
2.2. Evaluation of the FCSNP in the Capture of Peptides
Using UHPLC-Q-ToF and a mixture of two model peptides, the interaction with Cibacron blue present at the FCSNP was tested (Table 2
). The model peptides included a nonapeptide containing no hydrophobic region and alpha zein 34-mer with a long hydrophobic section. The nonapeptide was only obtained in the wash; this indicated that it was not captured by the molecular bait immobilized in the core of the nanoparticles. On the other hand, alpha zein34-mer was captured by the Cibacron blue [12
], and eluted with all three types of eluents, indicating that the peptide-FCSNP interactions were of varying hydrophobicity. The presence of alpha zein 34-mer in the wash was due to the excess of the amount of peptide incubated with the FCSNP. The peptides were found according to their characteristic mono or multi-charged ions, which were [M + H] and [M + 2H] for the nonapeptide and [M + 3H] for alpha zein34-mer, respectively. The experimental molecular mass of the peptides from washes and elutions coincided with their respective theoretical mass.
In the nonapeptide sequence, there are only three hydrophobic amino acids, proline, isoleucine, and valine (Table 2
, bold-faced print), and the hydrophobic interactions [12
] were not sufficiently strong to bind the molecular bait in the FCSNP. On the other hand, in the amino acidic sequence of the peptide alpha zein 34-mer, 20 of 34 amino acid residues were hydrophobic (Table 2
, bold-faced print), allowing strong, but varying, interactions with the molecular bait. Cibacron Blue is a pseudo-affinity molecular bait; therefore, it does not interact with proteins in a specific manner. However, it may interact with proteins with sufficiently long hydrophobic regions [19
] regardless of their mass.
2.4. FCSNP in the Exclusion of HMW Proteins and Capture of LMW Proteins
The simultaneous exclusion of HMW proteins by the polymeric shell and capture of LMW proteins by molecular bait (bifunctionality) by FCSNP was analyzed by SDS-PAGE. Nanoparticles were incubated with a reduced amount of protein (ten-fold reduction compared to the previous experiment), while maintaining the ratio (3.5 µg of each protein, BSA, myoglobin and aprotinin) for 60 min (Figure 6
). An excess of proteins were removed in the wash with PB (lane 2). Elution with 50% isopropanol obtained bands corresponding to BSA, as well as myoglobin and aprotinin (lane 3); this could indicate that these proteins were in excess or were interacting with molecular bait near the FCSNP surface. Thus, this elution could be considered as an intermediate step between washes and elutions.
Bifunctionality is evident in the elutions with 50% (v/v) methanol and ACN + NH4OH (lanes 4 and 5, respectively). Therefore, in order to obtain an optimal performance of the FCSNP, a lower amount of protein should be incubated to effectively remove HMW proteins present in the mixture and capture LMW proteins with a strong hydrophobic character.
Our results show the simultaneous exclusion of an HMW protein and capture of LMW proteins and peptides and coincide with those reported by Tamburro et al. [12
]. However, FCSNP showed a size three times smaller (243.9 ± 11.6 nm) than the particles reported by Tamburro et al. (700 nm). In addition, silica is a highly stable compound with low reactivity with biomolecules, and it can be easily functionalized with diverse compounds including molecular baits offering great potential for a variety of applications [29
]. Further, spherical shapes and narrow size distributions of silica nanoparticles can easily be reproduced [14
]. FCSNP also remained stable after twelve months of storage (data not shown).