**2. Seed-mediated growth toward hybrid nanostructures**

Wet-chemically seed-mediated growth provides an effective method for the synthesis of the hybrid nanoparticles with well-controlled structures, where the secondary species attach and sequentially grow on the preformed seeds. To ensure the deposited species welldispersed on the supports, heterogeneous nucleation and growth through atomic addition must be achieved and homogeneous nucleation should be avoided. Significant progress has

of the multifunctional hybrid nanomaterials.

contrast agents, and theranostic biomedical applications.

multimodal imaging and therapy are highlighted.

**2. Seed-mediated growth toward hybrid nanostructures** 

understanding of the interfacial interactions is therefore a significant basis for the fabrication

The functional inorganic hybrid nanomaterials with combined plasmonic-magnetic, plasmonic-fluorescent, or magnetic-fluorescent structures have unique optoelectronic properties for biomedical applications.[5, 8-10] The optical, electronic, magnetic properties could be controlled by adjusting their sizes, shapes, compositions, and surface chemistry. Several types of the hybrid nanostructures are potentially useful for biomedical applications.[5, 8] For example, the plasmonic-fluorescent materials would be interesting as dual-use biological tags, giving the ability to visualize labeled cells using both magnetic resonance and fluorescence imaging techniques, while external magnetic fields could be employed for the directed assembly of such materials.[11] In order to be compatible with biomedical environment, the surface of the nanohybrids is functionalized with appropriate amphiphilic polymer, silica, targeting ligand through chemisorption, covalent linkage, and ligand exchange.[12] After surface modification, the coated hybrid nanoparticles have the possibility of highly colloidal stabilization in aqueous media compatible with bioconjugation.[13] The delivery of the hybrid nanoparticles to targeted cells is an important and challenging approach in medical diagnosis and therapy. Development of biomoleculeconjugated strategies allows the hybrid nanoparticles delivering to targeted tissues through either antibody-antigen or ligand-receptor interactions.[12] Biomolecules, such as small molecules (vitamin, peptide, lipid, sugar) and larger ones (protein, DNA, antibody, enzyme, carbohydrate), are often used to conjugate onto the hybrid particles. Biomolecule-conjugated nanohybrid colloids have the possibility of highly selective binding with alive organ made them to be "biocompatible" behaviour and superior bioactivity at the supermolecular level.[5, 14, 15] These features have potentially applied in smart drug delivery vehicles,

This Chapter reviews the design, fabrication, and theranostic biomedical applications of the inorganic hybrid nanomaterials with combined plasmonic-magnetic, plasmonic-fluorescent, or magnetic-fluorescent structures. New collected properties of the hybrid nanostructures arising from the particle-particle interactions and the geometries are discussed from specific results of recent publications. The functionalizations of the hybrid nanostructures with amphiphilic polymer and silica coatings yielded water-soluble nanohybrid colloids are described. The subsequent conjugation of biomolecules with the coated nanohybrids afforded cellular targeting agents and the use of them as multimodal bioprobes for

Wet-chemically seed-mediated growth provides an effective method for the synthesis of the hybrid nanoparticles with well-controlled structures, where the secondary species attach and sequentially grow on the preformed seeds. To ensure the deposited species welldispersed on the supports, heterogeneous nucleation and growth through atomic addition must be achieved and homogeneous nucleation should be avoided. Significant progress has

**Figure 1.** A general sketch of reaction mechanisms for the formation of the hybrid nanostructures: (a-c) Heterogeneous nucleation and growth of secondary precursors on the preformed seeds; (d) Secondary precursors adsorbed on the opposite charged surfaced supports by photo-irradiation; (e) Reduced precipitation of secondary precursors on hydrophilic-surfaced supports; (f) Secondary precursor growth on the activated-surfaced supports; (g) One-pot self-controlled nucleation-growth.

been achieved in the synthesis of the core-shell and dumbbell nanoparticles combining two different components. When the individual components involved have similar crystal structures and lattice parameters, each component fuses together giving the dumbbell shape. In a dumbbell structure consisted of one particle-bounded another, charged transfer across nanoscale junction could significantly change local electronic configuration that give the remarkable properties. While large lattice space difference of the individual components results in the core shell-shaped structure obtained by growing a uniform layer of a shell material around colloidal particles. An isolation of core from surroundings could create the specific materials with emerged properties to those of the bare nanoparticles.[16, 17]

Functional Inorganic Nanohybrids for Biomedical Diagnosis 315

oxidation in 1-octadecene. The sizes (2-8 nm) of Au particles were controlled either by tailoring HAuCl4/oleylamine molar ratio or by controlling temperature at which HAuCl4 precursors was injected. The sizes (4-20 nm) of Fe3O4 particles were tuned by adjusting the Fe(CO)5/Au ratio. Hyeon el al.[20] synthesized metal/oxide core-shells (Au-Fe3O4, Ni- Fe3O4, Au-MnO, Pt-Fe3O4) through thermolysis of the mixtures of transition metal-oleate (Fe, Mn) complexes and metal-oleylamine (Au, Ag, Pt, Ni) complexes in oleylamine/octadecene. These complexes were prepared from transfer-phase reaction of the inexpensive metal salts and surfactants. Due to the high solubility of the prepared complex precursors in 1 octadecene solvent, this method was able to large-scale synthesis of the well-shaped nanohybrids per a singe preparation. The Au-Fe3O4 particles became soluble in water by encapsulating their surface with a PEG-phospholipid shell. The amino-functionalized Au-Fe3O4 nanohybrids conjugated with thiolated oligonucleotide sequences exhibited the red shift of the surface plasmon resonance of the gold particles and the enhanced signal intensity of the Fe3O4 particles in *T*2-weighted magnetic resonance imaging. These

conjugated hybrid nanostructures have potential in multimodal biomedical probes.

**Figure 2** shows plasmonic-magnetic Au-Co core-shell and Au-Ni spindly nanostructures synthesized by Li et al.[21] through one-pot solvothermal reaction of HAuCl4 and metal

**Figure 2.** One-pot protocol for plasmonic-magnetic hybrid nanostructures via noble-metal-induced reduction process. (a) Schematic illustration of the noble-metal-induced reduction process. TEM images of Au-Co core-shell (b) and Au-Ni spindly nanostructures (c). Reproduced with permission from ref.

[21]. Copyright 2010, American Chemical Society.

The colloidal nanohybrids are generated upon reaction of molecular precursors in solution in the presence of surfactants. Once the synthesis is activated at a suitable temperature, monomers are generated, and then induced nucleation of nanoparticles and sustain their subsequent enlargement. The organic surfactants play key roles along the courses of the hybrid formation. **Figure 1** shows general growth models for the fabrication of dumbbelland core shell-shaped hybrid nanostructures, that can be classified into the synthetic routes: (i) the direct heterogeneous growth of secondary precursors on the sites or tips of the preformed seeds (Figure 1 a-c); (ii) metal clusters adsorbed on the opposite-charged surface of the supports by photo-irradiation (Figure 1d); (iii) reduced precipitation of secondary precursors adsorbed on the hydrophilic-surfaced nanohybrids through ion-exchange deposition (Figure 1e); (iv) secondary precursor growth on the support surface after the chemical activation of their surface (Figure 1f); (v) one-pot growth of secondary precursors on the supports through self-assembled control (Figure 1g). Following these pathways, a large variety of the nanohybrids including metal-metal, metal-oxide, oxide-oxide, metalsemiconductor structures has been synthesized successively. In the following sections, we present types of the plasmonic-magnetic, plasmonic-fluorescent, and magnetic-fluorescent hybrid nanomaterials synthesized using seed-mediated growth methods along functionalization and subsequent bioconjugation of the hybrid nanostructures for biomedical applications.

### **2.1. Synthesis of plasmonic-magnetic hybrid nanostructures**

Plasmonic-magnetic nanohybrids provide a promising platform for developing optical and magnetic multifunctional probes for cell imaging applications. Materials researchers have recently been efforted to synthesize the hybrid nanostructures. Alivisatos et al.[18] synthesized gold-iron oxide core-shells via thermolysis of Fe(CO)5 at the surface of gold nanoparticles in octadecene/oleylamine/oleic acid. These hybrid nanostructures were formed by deposition of an iron shell around the gold core and subsequent oxidation of the metallic iron shell to form an iron oxide hollow through Kirkendall effect. The heterogeneous growth of iron precursors on the gold seeds was dependent on the molar ratio of oleylamine/oleic acid capping agents. The thinnest oxide shell (~2 nm) surrounded the gold nanoparticles was formed at oleylamine/oleic acid ratio of 1:1. Irregular polycrystalline iron oxide shells connected with the gold core were formed in the presence of oleylamine without oleic acid. With increasing the oleic acid concentration led to the iron nucleation and growth to be slowed because of the formation of stable iron oleate complex in high-temperature reaction. The gold-iron oxide core-shells exhibited the surface plasmon resonance shift of the gold particles and the magnetic hysteresis loop shift of the iron oxide particles, originating from the core-shells with particle-particle interactions. Sun et al.[19] synthesized Au-Fe3O4 dumbbells via thermolysis of Fe(CO)5 on Au particles and subsequent oxidation in 1-octadecene. The sizes (2-8 nm) of Au particles were controlled either by tailoring HAuCl4/oleylamine molar ratio or by controlling temperature at which HAuCl4 precursors was injected. The sizes (4-20 nm) of Fe3O4 particles were tuned by adjusting the Fe(CO)5/Au ratio. Hyeon el al.[20] synthesized metal/oxide core-shells (Au-Fe3O4, Ni- Fe3O4, Au-MnO, Pt-Fe3O4) through thermolysis of the mixtures of transition metal-oleate (Fe, Mn) complexes and metal-oleylamine (Au, Ag, Pt, Ni) complexes in oleylamine/octadecene. These complexes were prepared from transfer-phase reaction of the inexpensive metal salts and surfactants. Due to the high solubility of the prepared complex precursors in 1 octadecene solvent, this method was able to large-scale synthesis of the well-shaped nanohybrids per a singe preparation. The Au-Fe3O4 particles became soluble in water by encapsulating their surface with a PEG-phospholipid shell. The amino-functionalized Au-Fe3O4 nanohybrids conjugated with thiolated oligonucleotide sequences exhibited the red shift of the surface plasmon resonance of the gold particles and the enhanced signal intensity of the Fe3O4 particles in *T*2-weighted magnetic resonance imaging. These conjugated hybrid nanostructures have potential in multimodal biomedical probes.

314 Practical Applications in Biomedical Engineering

biomedical applications.

results in the core shell-shaped structure obtained by growing a uniform layer of a shell material around colloidal particles. An isolation of core from surroundings could create the

The colloidal nanohybrids are generated upon reaction of molecular precursors in solution in the presence of surfactants. Once the synthesis is activated at a suitable temperature, monomers are generated, and then induced nucleation of nanoparticles and sustain their subsequent enlargement. The organic surfactants play key roles along the courses of the hybrid formation. **Figure 1** shows general growth models for the fabrication of dumbbelland core shell-shaped hybrid nanostructures, that can be classified into the synthetic routes: (i) the direct heterogeneous growth of secondary precursors on the sites or tips of the preformed seeds (Figure 1 a-c); (ii) metal clusters adsorbed on the opposite-charged surface of the supports by photo-irradiation (Figure 1d); (iii) reduced precipitation of secondary precursors adsorbed on the hydrophilic-surfaced nanohybrids through ion-exchange deposition (Figure 1e); (iv) secondary precursor growth on the support surface after the chemical activation of their surface (Figure 1f); (v) one-pot growth of secondary precursors on the supports through self-assembled control (Figure 1g). Following these pathways, a large variety of the nanohybrids including metal-metal, metal-oxide, oxide-oxide, metalsemiconductor structures has been synthesized successively. In the following sections, we present types of the plasmonic-magnetic, plasmonic-fluorescent, and magnetic-fluorescent hybrid nanomaterials synthesized using seed-mediated growth methods along functionalization and subsequent bioconjugation of the hybrid nanostructures for

specific materials with emerged properties to those of the bare nanoparticles.[16, 17]

**2.1. Synthesis of plasmonic-magnetic hybrid nanostructures** 

Plasmonic-magnetic nanohybrids provide a promising platform for developing optical and magnetic multifunctional probes for cell imaging applications. Materials researchers have recently been efforted to synthesize the hybrid nanostructures. Alivisatos et al.[18] synthesized gold-iron oxide core-shells via thermolysis of Fe(CO)5 at the surface of gold nanoparticles in octadecene/oleylamine/oleic acid. These hybrid nanostructures were formed by deposition of an iron shell around the gold core and subsequent oxidation of the metallic iron shell to form an iron oxide hollow through Kirkendall effect. The heterogeneous growth of iron precursors on the gold seeds was dependent on the molar ratio of oleylamine/oleic acid capping agents. The thinnest oxide shell (~2 nm) surrounded the gold nanoparticles was formed at oleylamine/oleic acid ratio of 1:1. Irregular polycrystalline iron oxide shells connected with the gold core were formed in the presence of oleylamine without oleic acid. With increasing the oleic acid concentration led to the iron nucleation and growth to be slowed because of the formation of stable iron oleate complex in high-temperature reaction. The gold-iron oxide core-shells exhibited the surface plasmon resonance shift of the gold particles and the magnetic hysteresis loop shift of the iron oxide particles, originating from the core-shells with particle-particle interactions. Sun et al.[19] synthesized Au-Fe3O4 dumbbells via thermolysis of Fe(CO)5 on Au particles and subsequent **Figure 2** shows plasmonic-magnetic Au-Co core-shell and Au-Ni spindly nanostructures synthesized by Li et al.[21] through one-pot solvothermal reaction of HAuCl4 and metal

**Figure 2.** One-pot protocol for plasmonic-magnetic hybrid nanostructures via noble-metal-induced reduction process. (a) Schematic illustration of the noble-metal-induced reduction process. TEM images of Au-Co core-shell (b) and Au-Ni spindly nanostructures (c). Reproduced with permission from ref. [21]. Copyright 2010, American Chemical Society.

nitrate (Co(NO3)2·6H2O or Ni(NO3)2·6H2O) in octadecylamine used as both solvent and surfactant. The formation of the hybrid structures was illustrated by metal-induced reduction process namely, octadecylamine-supplied electron cloud surrounded Au atoms reduced transition metal ions to form the nanohybrids. The strong magnetism of the plasmonic-magnetic nanohybrids and the CO superior catalytic activity of the Au-Co coreshells were showed as a result of incorporating magnetic heterometals into gold particles. The authors [22] also synthesized bifunctional Au-Fe3O4 nanohybrids through the conjugation of Au particles to thiol-modified Fe3O4 nanoparticles. Lysine contained both amino and carboxylic groups played dual roles as both linker and capping agent in attaching metals on Fe2O3 particles. The hybrid nanostructures can be used for magnetic separation of biosubstances and for protein separation. The authors [27] also synthesized hexagonal pyramid-like shaped Au-ZnO nanohybrids based on the growth of ZnO particles derived from a mixture of zinc acetate/oleylamine/dodecanol on Au seeds. The nanopyramids composed of Au particles as the tip and hexagonal ZnO pyramid as the tail. The Au-ZnO hybrid nanopyramids with homogeneous composition and controlled morphology showed better photocatalytic efficiency than ZnO nanocrystals.

Functional Inorganic Nanohybrids for Biomedical Diagnosis 317

**Figure 3.** Growth of Au shell around pyramidal CdSe nanocrystals. (a) Scheme of the Au growth process onto CdSe nanocrystals. (b) Incubation of CdSe with Au-tetra-*n*-butylammonium

borohydride/dodecyltrimethylammonium bromide led to Au clusters positioned at apexes of the CdSe pyramids. (c) STEM image of Au/CdSe hybrids with sharp angles as reactive sites for the nucleation of Au particles. Reproduced with permission from ref. [24]. Copyright 2010, The Royal Society of Chemistry.

on the corners and tips of the CdSe nanocrystals through the suitable choice of the metal precursor and the surface ligand concentration. The pyramid structure provided relatively sharp angles which were highly reactive sites for the nucleation of the gold particles. Banin et al.[25] also synthesized cage-shaped Ru/Cu2S nanohybrids by adding Ru(acac)3 to Cu2S seed suspension in octadecylamine. Metallic Ru selectively grew on the crystal edges of the Cu2S nanocages to form a symmetrical cage around a Cu2S core. The cage formation could be due to the capping of thiol ligands on the Cu2S facets to block the growth of these crystal facets. These Ru/Cu2S nanocages were used as an excellent electrocatalyst for H2O2 sensing

The recent progress made in the synthesis of magnetic-fluorescent nanohybrids with combined magneto-transport properties has been reported. Cozzoli et al.[26] reported the two-step seeded-growth for the selective synthesis of Co-tipped CdSe/CdS core-shell nanorods. A two-step procedure consisted of injection of tri-n-octyl phosphine sulphide/CdSe seeds to Cd-surfactant complexes and heated at 350-380 °C to form CdSe/CdS core-shells. Co clusters were then attached with the CdSe/CdS seeds by injecting Co2(CO)8 solution into octadecene solvent containing CdSe/CdS seeds at 200-240 °C under inert atmosphere. An excess volume of oleic acid was added to the growing mixture to stabilize the hybrid nanostructures formed after reaction completion. The formation of the

with catalytic activity higher than Cu2S electrode.
