Abstract In an effort to prepare efficient and uniform biolabel, a kind of novel fluorophore doped silica nanoparticles via water-in-oil (W/O) microemulsion method has been developed. 5-aminofluorescein (AMF) was connected to 3-aminopropyltriethyloxysilane (APTEOS) with dianhydride of diethylenetriaminepentaacetic acid (DTPAA) to form a monomer fluorophore silica precursor. The controlled co-hydrolysis of tetraethyl orthosilicate (TEOS) and the precursor in W/O microemulsion leads to the formation of monodisperse fluorophore doped silica nanoparticles. The transmission electron microscope (TEM) photograph showed that the particles are very uniform and the fluorophores are dispersed homogeneously throughout the silica network, with a diameter of about 40nm.The nanoparticles have shown several unique advantages over the previous nano-biolabel in easy preparation, good photostability and significantly reduced fluorescent dye leaching. The particles are potential of good biocompatibility, since they have a silica surface and can thus be modified easily and connected to various biomolecules for added biochemical functionality.
Keywords 5-aminofluorescein, microemulsion, fluorescent nanoparticles

1 INTRODUTION
Recently, molecular probes for bimolecular recognition are of great importance in the fields of chemistry, biology and medical science as well as in biotechnology. Fluorescent labeling of biological materials using small organic dyes has been widely employed in the life science including diagnostics and biological imaging ^[1]. Problems arise mainly from poor photostability and brightness, especially for samples with high background fluorescence ^[2]. Therefore, it is highly desirable to develop new fluorescent probes for biochemical assays. Fluorophore doped nanoparticles have draw much more attention recently ^[3,4] since the fluorophores doped inside the nanoparticles are well protected from the surrounding environments and are, thus, immune to the potential quenching and bleaching in solution. Recently, Yang et al ^[5] reported the use of lissamine rhodamine B sulfonylchloride hybrid silica nanoparticles in the sandwich-model immunoassay for the detection of trace level HBsAg. They coupled the dye fluorescein isothiocyanate (FITC) into the silica spheres in a quite rigorous condition because the organic dye molecules could not be easily doped into the silica matrix. One of the reasons is that many dyes are hydrophobic, which will be quickly partitioned to the organic phase during the polymerization of tetraethyl orthosilicate (TEOS). Even for highly water-soluble dyes, a serious dye leakage from the silica matrix after the dispersion in an aqueous solution still exists if there are no retaining forces between dye molecules and the silica matrix ^[6]. It has been reported the dye leaching can be reduced if the dye has been connected to IgG, a Y shape protein ^[7], but IgG is very expensive, which leads to limit in practice.
    In the present work, AMF was coupled with DTPAA and APTEOS. By synthesizing a monomer fluorophore silica precursor of 5-aminofluorescein (AMF), we have successfully prepared fluorescent hybrid silica nanoparticles via water-in-oil (W/O) microemulsion method. The particles have uniform size, good photostability, good biocompatibility, and high quantum efficiency. Furthermore, it can significantly reduce fluorescent dye leaching from the nanoparticles to use the precursor as core material, since DTPAA is a dianhydride compound with polycarboxyl that leads to the high stability of the precursor. The silica shell surface can be further functionalized in numerous ways to meet different needs.

2 EXPERIMENTAL
2.1 Materials and apparatus
5-aminofluorescein (AMF, Acros), 3-aminopropyltriethyloxysilane (APTEOS, Acros), tetraethyl orthosilicate (TEOS, Aldrich), dimethylsulfoxide (DMSO), Triton X-100, n-hexanol, and cyclehexane, ammonium hydroxide (28-30wt %) were used as received, dianhydride of diethylenetriaminepentaacetic acid (DTPAA) prepared as the reference ^[8], Distilled water was used throughout.
    Fluorescence was determinated with a Hitachi F-4500 fluorescence spectrophotometer. Size and shape of the nanoparticles were characterized with Philip Tecnai-10 transmission electron microscope.
2.2 preparation methods
2.2.1 Synthesis of monomer fluorophore silica precursor
Monomer fluorophore silica precursor, AMF-DTPAA-APTEOS (abbreviate as ADA), was prepared by the following procedure:

    DTPAA (0.14mmol) was dissolved in 2ml DMSO, and then AMF (0.14mmol) was added with stirring, after stirring for 8h, 33mL APTEOS (0.14mmol) was added. The mixture was allowed to react overnight at ambient temperature. Since the byproduct did not affect the following procedure and could be easily removed after formation of the nanoparticles, the monomer precursor was not further isolated.

3 RESULTS AND DISCUSSION
3.1 The preparation of nanoparticles
The nanoparticles have been prepared by the water-in-oil microemulsion method. A controlled co-hydrolysis of TEOS and the monomer precursor in W/O microemulsion has led to the formation of monodisperse ADA-doped silica nanoparticles. The ADA-doped nanoparticles are uniform in size, 40±5nm in diameter, as characterized by TEM (Fig.1).
    Fluorescence spectrometer measurements were also used to characterize the nanoparticles. The excitation and the emission spectra of AMF, ADA monomer precursor and ADA-doped nanoparticles were measured in aqueous solution; AMF shows emission at 519nm when excited at 496nm excitation band maxima (Fig.2). There is a blue shift of 6nm in excitation spectra of ADA compared with that of AMF, while ADA-doped nanoparticles have a red shift. The blue shift of ADA may be due to the formation of chemical bond between AMF and DTPAA, while the red shift of nanoparticles may be due to the presence of silica network surrounding the fluorophores.

3.2 Dye leaching experiment
To date, most of the fluorescent material that has been used to prepare core-shell nanoparticles is limited in metal coordination compound; while organic fluorescent labels that have higher quantum efficiency have not been widely used in the preparation of core-shell nanoparticles. Experiments show that using traditional method of preparing core-shell nanoparticles to prepare organic dye doped core-shell nanoparticles induce remarkable fluorescent dye leakage. Dye leakage could be reduced by connecting the dye molecule to some protein with unique shape, such as IgG. Because IgG has a "Y" shape organized protein structure, which can increase the steric hindrance and protected the dye leaching from the nanoparticles. However, IgG is very expensive, which leads to limit in practice. IgG is also prone to change its shape after denaturalization which may cause addition leakage of the dye. In this paper, forming chemical bond among AFM, DTPAA, APTEOS and the silica framework prevented leakage of the fluorescein. It is obvious that the present method is more stable and much less expensive; the result is shown in Table 1. After washing for 9 times, the fluorescence intensity dropped only 5% of the initial intensity, which may result from the departing of minor unreacted byproduct in the precursor solution. Fluorescence intensity did not show visible change after washing for 5 times, which indicated the dye molecular has been firmly attached to the silica framework by the chemical bond.

4 CONCLUSIONS
The present approach of making fluorophore-doped nanoparticles is a general one. There are several important advantages of these nanoparticles with functionality for biomolecular label. The nanoparticles are easily prepared using relatively simple procedures; they have uniform size and good photostability. And most important, this approach gives a satisfactory result to dye leakage. Surface of the fluorophore-doped silica nanoparticles can be functionalized in different ways to meet the needs on the basis of a variety of biomolecular recognition mechanisms. The versatility makes, in our opinion, ADA-doped nanoparticles a good choice as markers for biochemical assays.