ABSTRACT
For the visualization of biologically relevant molecules and activities inside living cells and early detection of diseases, semiconductor quantum dots offer high-quality optical imaging and various particle-cell immobilization advantages than organic markers. But the toxic ligands used in most of the synthesis processes and the cytotoxicity due to the release of heavy metals are the major issues. ZnO is a good candidate to overcome these issues due to its biocompatibility and excellent optical properties. However, the required conditions for bioimaging are not simultaneously satisfied by ZnO due to one of the following problems: high temperature or too fast chemical reaction, leading to surface defects, resulting in poor optical properties; no suitable surface capping, leading to particle agglomeration in water or uncontrolled particle shape; toxic ligands used in the process, leading to post-treatment and potential contamination/toxicity
of the final colloidal solution; and no suitable doping, leading to only ultraviolet (UV) emission, not detectable by common confocal microscopy. In this chapter, we report the synthesis and surface modification of biofriendly ZnO-based colloids, which have been tested both in vitro and in vivo in human tumor cells and rat models, providing significant advantages on quantum confinement effects, superior optical properties, nontoxicity, and a unique dual-color imaging feature. 12.1 Introduction
Advances in molecular medicines require the optimum detection of individual biomolecules, cell components, and other biological entities. Traditional fluorescent dyes (e.g., fluorescein, ethidium, methyl coumain, rhodamine, etc.)1 have a number of physical and chemical limitations that include low photostability, narrow absorption bands, broad emission spectra, and differences in chemical properties of the dyes, making multiple, parallel assays impractical.2 Fluorescent semiconductor (groups II-VI) nanocrystals, often referred to as quantum dots (QDs), have unique optical and electrical properties, such as size-and compositiontunable fluorescence emission from visible to infrared wavelengths, large absorption coefficients across a wide spectral range, and a very high level of brightness and photostability.3 The major issue of Cd-containing QDs is the potential cytotoxicity; therefore, alternative materials that do not contain cadmium and are more biocompatible are required.4 ZnO is a versatile semiconductor with a wide bandgap (~3.37eV) and an extremely large excitonbinding energy (60 meV), which makes the exciton state stable at room temperature and above. Its unique optical properties and biocompatibility are huge advantages as a better candidate for bioimaging than metals and chalcogenide (S, Se, Te) nanoparticles. However, research on ZnO for bioapplications considerably lags behind the other candidates because the synthesis is not as well developed and the doping and surface modification of ZnO are less well understood. A few methods for obtaining ZnO nanocrystals in aqueous solution at low temperature have been reported, but they all involve strong alkaline media5-8 or annealing9, and, therefore, are not suitable for bioapplications. To make ZnO QDs for in vivo and
in vitro bioapplications, the synthesis process has to meet several requirements: (1) only contains biocompatible materials, (2) uses a suitable surface-capping agent to ensure nanosized ZnO particles in a stable colloidal solution, (3) provides chemical functional groups on the ZnO surface that will eventually bind to biomolecules, and (4) ensures photostability and efficient fluorescence of the ZnO nanocrystals. A biofriendly synthesis method for ZnO using the buffer tris(hydroxymethyl)aminomethane was reported10, but no actual biotest was conducted. A surface modification method using an organosilane cross-linker was reported11, but only an organic dye was tested as an indication for further biomolecular attachment. Recently, we reported the preliminary study of bioimaging on plant cells by ZnO.12,13 Since ZnO intrinsically emits in the UV wavelength, doping with suitable elements is an effective method to adjust their electrical, optical, and magnetic properties, which is crucial for its practical applications. After bandgap modification, ZnO could provide both UV and blue-violet emissions, which fill up the missing spectra range of current QDs. Doping of ZnO by Mg, Ni, Cu, In, and Al has been reported to improve the electrical conductivity.14-17 Doping of ZnO by Mn, Ni, Cu, and Co has been reported to improve ferromagnetic properties.18-21 Some other dopants such as Li, Na, and K were also reported22-23, but due to their small atomic radii, these elements occupy the interstitial sites, rather than substitution sites, inducing strain and increasing the formation of native defects (vacancies). For bioimaging applications, the preferred dopants should have similar atomic radii with Zn and can reduce the bandgap of ZnO to enhance photoluminescence (PL) emission in visible wavelengths. We need to avoid surface defects, have a controllable bandgap, and have strong and stable PL emission. It is an added value if one marker can label both the nucleus and the cytoplasm of a cell simultaneously. This eliminates the complexity of two-step labeling using two different markers and the need of two excitation sources in the case of fluorescent dyes. The chemical synthesis and surface modification of ZnO nanocrystals have been reported by us recently.24,25 Many different types of surface-capping agents have been studied, including 3-aminopropyl trimethoxysilane (Am), mercaptosuccinic acid (Ms), 3-mercaptopropyl trimethoxysilane (Mp), and polyvinylpyrrolidone (Pv); two types of aminosilanes,
aminoethyl aminopropyl trimethoxysilane (Z60) and aminoethyl aminopropylsilane triol homopolymer water solution (Z61); and titania (TiO2) and silica (SiO2) through the sol-gel route. Bandgap modification of ZnO has been achieved by doping ZnO nanocrystals with Co, Cu, and Ni cations in combination with surface capping. The applications of these nanoparticles on bioimaging of both human cells and plant systems were tested and proven. Due to the small size, the double amino surface functional groups, and the high PL emission intensity, ZnO nanoparticles showed dual-colored images with blue emission at the nucleus and turquoise emission at the cytoplasm simultaneously. Cytotoxicity was tested on human osteosarcoma cells, which proved the nontoxicity of the Z60-and Z61-capped ZnO nanocrystals. The maximum inhibitory nanoparticle loadings corresponding to 50% cell viability (IC50) of the synthesized ZnO were compared to commercial CdSe/ZnS QDs. The quantum yields (QYs) of our nanocrystals varied from 79% to 95%, which are higher than most of the current Cd-based QDs (typically 25-30%).26 12.2 Particle Size Control through Chemical
Synthesis and Surface ModificationsTo visualize the biological details in the living cells and the nucleus, the particle size of the biomarker must be below a few nanometers. Therefore, the control of particle size in an aqueous system is crucial. Both the synthesis parameters and the surface-capping method are important control factors for particle size. In the current study, ZnO colloidal solutions were synthesized by refluxing zinc acetate dehydrate and Zn(Ac)2·2H2O methanol in a molar ratio of 0.03:4 at 66-67°C for six to seven hours. Nickel, cobalt, and copper dopants were added with different molar ratios (x = 0.05, 0.1, 0.15, 0.2) during the synthesis. A capping agent was added after cooling the reactants in ice water. The reaction time and temperature, fast cooling, and proper surface modification by the above-described capping agents are the key parameters in controlling the particle size. Figure 12.1 shows the field-emission scanning electron microscope (FESEM) images of (a) Z60-capped 5%Ni-doped ZnO particles precipitated on an AAO template, the particle size smaller than 10 nm, (b) uncapped 5%Ni-doped ZnO particles in different sizes from submicron to 1.5 μm, (c) uncapped Cu-doped ZnO particles, and (d)
uncapped pure ZnO particles in irregular shape and different sizes. These images indicate that capping is very effective in controlling the particle size; without capping, the particles grow to a larger size with a wider size distribution. It is noted that Ni-doped ZnO grew to a cabbage-like shape with perfect circular outlines, but uncapped pure ZnO grew to irregular shapes. This means that the dopant changes the lattice structure and facial energies of the ZnO, leading to preferential shapes. Similar shape and surface structures were observed in Co-doped ZnO particles (not shown here), while Cu-doped particles only grew into spherical particles without surface structures, as shown in Fig. 12.1c. Figure 12.2 shows the particle size distribution curve of a typical effectively capped ZnO colloid synthesized by this study, measured by Zetasizer (Malvern Nano ZS) using the 5%CoZnO-Z60 colloidal sample (7 days after synthesis). The size is between 1 to 2 nm with 97% particles between 1.5 to 2 nm.
