| Fluorescence probes have been widely used in biomedical areas, including imaging, sensing and diagnosis. The fluorescence property is certainly a basic requirement for the fluorescence probes, but the bio-safety of the probes is more crucial for their biomedical applications. Organic dyes are the first generation of fluorescence probes, and they are still widely used nowadays. But due to severe problems including stability and toxicity, the widely used organic dyes in biomedical research are being gradually replaced by new types of nanoprobes, especially quantum dots (QDs). QDs have superior fluorescence properties, such as narrow emission spectra, strong fluorescence and high resistance to photo-bleaching, hence have been extensively studied. However, the toxicity of their heavy metal ions is a big concern, though encapsulation by silica or polymers may reduce the risk of heavy metal ion release.Green fluorescence protein (GFP), originally discovered from jellyfish, has been engineered into a set of fluorescent proteins (FPs) with a wide spectrum of emission wavelength and excellent fluorescence properties. The high quantum yield and stable fluorescence performance make FPs possible to be used even for single molecule detecting. However, most application of FPs carried out at gene level. Purified FPs are not suitable as the direct fluorescence probes due to their instability against protease, denaturant and many other factors.The aim of this work is to develop a new type of fluorescence probes with good bio-safety and great fluorescence properties. The basic idea is to encapsulate and protect the bio-safe FPs into silica NPs.We have developed two methods to encapsulate GFP in silica NPs:1) Non-covalent encapsulation method The encapsulation of proteins in biocompatible silica nanoparticles (NPs) has been extensively studied for many applications, such as biosensor, bioreactor, imaging and drug-delivery. Unfortunately, a recent study has shown that non-covalent encapsulation of a protein in silica NPs is critically depended on the pI value of the protein. Negatively-charged proteins (pI<7) are difficult to be encapsulated and easy to leak out of silica, because of the repulsion of the negative charges on silica NPs.To overcome this problem, we added a His-tag to the C-terminal of the enhanced green fluorescence protein (EGFP) is negatively-charged (pI =5.99), and modified the widely-used conventional reverse microemulsion method by simply adding a small amount of calcium ions. This new procedure has been proved to be efficient for negatively-charged proteins, and has been successfully applied to encapsulate other negatively-charged proteins and peptides. The efficient entrapping of EGFP in silica NP is due to the coordination between the His-tag of EGFP and the immobilized calcium ions.We found that the silica encapsulation can significantly improve the stabilities and fluorescent properties of EGFP, showing its potential application for bio-imaging.2) Covalent methodSince non-covalent encapsulation cannot avoid the protein leaching problem, EGFP non-covalently encapsulated in silica NPs is not ideal for the long-term use. Therefore, we developed a simple and ultra-efficient covalent method to entrap EGFP in silica NP.To covalently entrap EGFP in silica NP, we pre-treated EGFP by attaching (3-aminopropyl) trimethoxysilane (APTS) groups to–COOH groups on the surface of EGFP. The APTS-EGFP was then encapsulated in silica NP by the common reversible (water-in-oil) emulsion method. Comparing to the non-covalent one, the covalently-entrapped EGFP shows better fluorescence and stabilities against protease, denaturant, heat and storage. The remarkably improved fluorescence property and stabilities make this EGFP-encapsulated silica NP a robust and safe fluorescence probe. |
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