The encapsulation of proteins in biocompatible silica nanoparticles (NPs) has been extensively studied for many applications, such as biosensors, bioreactors, imaging, and drug delivery. 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. 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 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