The enhanced green fluorescence protein (EGFP) is efficiently encapsulated in silica nanoparticle by multiple covalent bonds. The silica encapsulation greatly increases EGFP's fluorescence intensity and stabilities against protease, denaturant and heat, making it a potential fluorescence probe for cellular imaging.
The encapsulation of proteins in biocompatible silica nanoparticles (NPs) has been extensively studied for many applications, such as biosensors, bioreactors, imaging, and drug delivery. [1][2][3][4][5][6][7][8][9][10][11][12] Unfortunately, a recent study has shown that the encapsulation of a protein in silica NPs is critically dependent on the pI value of the protein.[3] Negatively charged proteins (pI < 7) are difficult to encapsulate and escape easily because of repulsion with the negative charges on the silica NPs. Herein, we report a facile method to encapsulate proteins, including negatively charged proteins. We show that His-tagged enhanced green fluorescent protein (EGFP; His tag = polyhistidine; pI = 5.99) can be easily and stably encapsulated in silica NPs by using the widely used reverse-microemulsion method [7] with a small amount of additional calcium ions. The remarkably improved fluorescence properties and stability make this EGFP-encapsulated silica NP a robust and safe fluorescence probe.Scheme 1 shows the modified reverse-microemulsion method for encapsulating His-tagged proteins. The added Ca 2+ ions form ionic bonds with the oxygen atoms on the silica NPs, and provide anchors to link His-tagged proteins to the silica shell through coordinate bonds between the Ca 2+ ions and the histidine residues of the His-tagged proteins.In the presence of Ca 2+ ions, a significant amount of EGFP can be encapsulated in silica NPs. The TEM image of EGFP-encapsulated silica NPs in the presence of Ca 2+ ions (denoted as EGFP-Ca@SiO 2 ) shows an apparently hollow center (Figure 1 a, and see the Supporting Information). However, in the absence of Ca 2+ ions, the silica NPs appear as solid spheres (Figure 1 b). X-ray diffraction (XRD) analysis indicated that silica NPs, which were prepared either in the presence or absence of Ca 2+ ions, are amorphous (see the Supporting Information). Elemental analysis shows that there is 4.195 % nitrogen in EGFP-Ca@SiO 2 , which corresponds to 24.8 % EGFP (nitrogen content in EGFP is 16.9 %) in the NPs. The calcium content is about 0.4 %, as determined by Zeeman atomic absorption spectrometery. The coordinate bonds between the Ca 2+ ions and the His tags are essential for encapsulating negatively charged proteins in the silica NPs. A control experiment with FITC-labeled (FITC = fluorescein isothiocyanate) bovine serum albumin (BSA; pI = 4.7) without the His tag showed little encapsulation in the silica NPs, even in the presence of Ca 2+ ions (data not shown). When Scheme 1. Reverse-microemulsion procedure for encapsulating Histagged proteins. EGFPs (green) with His tags (red) are anchored to the silica shell through coordinate bonds between Ca 2+ ions (yellow) and the histidine residues of the His tags. TEOS = tetraethoxysilane.
A simple method is used to covalently encapsulate enzymes in silica nanoparticles. The encapsulation is highlighted by the high enzyme loading and porous channels that provide efficient diffusion for small substrate and product molecules while preventing protease degradation.
The encapsulation of proteins in biocompatible silica nanoparticles (NPs) has been extensively studied for many applications, such as biosensors, bioreactors, imaging, and drug delivery. [1][2][3][4][5][6][7][8][9][10][11][12] Unfortunately, a recent study has shown that the encapsulation of a protein in silica NPs is critically dependent on the pI value of the protein.[3] Negatively charged proteins (pI < 7) are difficult to encapsulate and escape easily because of repulsion with the negative charges on the silica NPs. Herein, we report a facile method to encapsulate proteins, including negatively charged proteins. We show that His-tagged enhanced green fluorescent protein (EGFP; His tag = polyhistidine; pI = 5.99) can be easily and stably encapsulated in silica NPs by using the widely used reverse-microemulsion method [7] with a small amount of additional calcium ions. The remarkably improved fluorescence properties and stability make this EGFP-encapsulated silica NP a robust and safe fluorescence probe.Scheme 1 shows the modified reverse-microemulsion method for encapsulating His-tagged proteins. The added Ca 2+ ions form ionic bonds with the oxygen atoms on the silica NPs, and provide anchors to link His-tagged proteins to the silica shell through coordinate bonds between the Ca 2+ ions and the histidine residues of the His-tagged proteins.In the presence of Ca 2+ ions, a significant amount of EGFP can be encapsulated in silica NPs. The TEM image of EGFP-encapsulated silica NPs in the presence of Ca 2+ ions (denoted as EGFP-Ca@SiO 2 ) shows an apparently hollow center (Figure 1 a, and see the Supporting Information). However, in the absence of Ca 2+ ions, the silica NPs appear as solid spheres (Figure 1 b). X-ray diffraction (XRD) analysis indicated that silica NPs, which were prepared either in the presence or absence of Ca 2+ ions, are amorphous (see the Supporting Information). Elemental analysis shows that there is 4.195 % nitrogen in EGFP-Ca@SiO 2 , which corresponds to 24.8 % EGFP (nitrogen content in EGFP is 16.9 %) in the NPs. The calcium content is about 0.4 %, as determined by Zeeman atomic absorption spectrometery. The coordinate bonds between the Ca 2+ ions and the His tags are essential for encapsulating negatively charged proteins in the silica NPs. A control experiment with FITC-labeled (FITC = fluorescein isothiocyanate) bovine serum albumin (BSA; pI = 4.7) without the His tag showed little encapsulation in the silica NPs, even in the presence of Ca 2+ ions (data not shown). When Scheme 1. Reverse-microemulsion procedure for encapsulating Histagged proteins. EGFPs (green) with His tags (red) are anchored to the silica shell through coordinate bonds between Ca 2+ ions (yellow) and the histidine residues of the His tags. TEOS = tetraethoxysilane.
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