## 2 Preparation of Magnetic-Fluorescent Nanocomposites
### Fluorescent Silica Coated Magnetic Nanoparticles  
Different strategies have been followed to produce a reliable fluorescent nanostructure. One of the most used and most straightforward approaches to avoid the quenching to the fluorescent entity is to coat the magnetic core with a layer of SiO\({}_{2}\). Through careful control of the thickness of the silica shell, the quenching of the fluorophore by the magnetic core may be prevented. Furthermore, silica can be easily functionalized, facilitating the bonding of fluorescent molecules to the surface. Many different strategies following this approach have been reported in the last years. Silica coated nanoparticles have been functionalized using different organic dyes. [PERSON] et al. [19] reported the preparation of FITC-labelled \(\gamma\)-Fe\({}_{2}\)O\({}_{3}\)-SiO\({}_{2}\)-AP nanoparticles. FITC (Fluorescein 5-isothiocyanate) is a fluorescent dye that is pH dependent. For this dye, the fluorescence is stronger at pH between 5 and 8, allowing the use of FITC functionalised nanoparticles as pH-sensing fluorescent probes in imaging (Figure 1). Another strategy to develop fluorescent magnetic nanostructures is to incorporate the fluorescent entity directly into the silica shell. The production of monodisperse and size-controllable core-shell nanoparticles in which FITC was incorporated in the nonporous silica shell has been reported a number of times [20; 21; 22]. The silica coated magnetic nanoparticles with organic dyes such as FITC can be further modified with folic acid and pH-sensitive polymers such as poly(2-hydroxyl  
Figure 1: Labelling of \(\gamma\)-Fe\({}_{2}\)O\({}_{3}\)–SiO\({}_{2}\)-AP and \(\gamma\)-Fe\({}_{2}\)O\({}_{3}\)–SiO\({}_{2}\)-AP–CMCS nanoparticles with FITC. Adapted from ref. [19]. methacrylate) (P(HEMA)) [23], poly(acrylic acid) (PAA) [24] or amino acids such as L-lysine ad L-arginine [25]. Rhodamine B isothiyanate (RITC) has also been used as fluorescent organic dye in the synthesis of fluorescent silica coated magnetic nanoparticles. For example, [PERSON] et al. reported the incorporation of magnetic iron nanoparticles and the organic dye into a silica coating of nanoparticles with further conjugation with Cetuximab for targeting and imaging cancer cells [26]. In another study, squatyliumindocyanine (Cy5-NH\({}_{2}\)) was used as a dye to functionalise Fe\({}_{3}\)O\({}_{4}\)@SiO\({}_{2}\) nanoparticles with an outer shell of carboxymethyl chitosan (CMCS) [27]. The incorporation of fluorescent QDs to the silica shell in silica coated magnetic nanostructures has also been investigated and reported. QDs have unique optical properites and this makes them an interesting material for bioimaging or biolabelling. However, the potential leakage of heavy metals is a serious drawback for their use in biomedical applications. Coating QDs in a biocompatible material such as silica has arisen as an option to prevent the leaking. For example, CdSe QDs were used in the synthesis of multifunctional fluorescent hollow silica coated magnetic nanoparticles in which both materials were trapped in a silica shell. Folic acid was conjugated to silica nanocomposites to be used for tumour cell imaging [28]. [PERSON] et al. prepared Fe\({}_{3}\)O\({}_{4}\)@SiO\({}_{2}\)-SH nanoparticles via the hydrolysis of a sol-gel precursor. In the last synthetic step, Fe\({}_{3}\)O\({}_{4}\)@SiO\({}_{2}\)-SH were combined with a water soluble CdSe/ZnS QDs solution, resulting in the final fluorescent material [17]. Magnetic-encoded fluorescent CdTe/Fe\({}_{3}\)O\({}_{4}\)@SiO\({}_{2}\) nanospheres were synthesized following a reverse microemulsion method (Figure 2) [29]. The same method was used by [PERSON] et al. in the development CdTe-Fe\({}_{3}\)O\({}_{4}\)@SiO\({}_{2}\) microspheres which were covalently bonded to graphene oxide [30]. The reverse microemulsion method was also chosen for the preparation of fluorescent magnetic nanoparticles consisting of silica wrapped CdTe and Fe\({}_{3}\)O\({}_{4}\) nanoparticles conjugated with anti-BRCAA1 antibody.