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Autor: anton • March 8, 2011 • 2,150 Words (9 Pages) • 959 Views
Synthesis of Nanoparticles for Bioimaging
The goal was to prepare nanoparticles in microemulsions. The experimental procedure was learned by preparing silica and gold nanoparticles. Particle characterization was done using Dynamic Light Scattering. The size of the silica and gold nanoparticles was found to decrease with increased w/o. The aim of this experiment was to synthesis fluorescent nanoparticles and gold nanoparticles. These are commonly used in bioimaging.
As researchers continue to develop a wide range of nanoparticle-enabled technologies for bioimaging, there is a growing need to understand how the basic physical properties of a particular nanoparticle affect biologically relevant behavior such as cellular uptake.
The most popular microemulsion system for preparation of nanoparticle is Ð²Ð‚Ñšwater-in-oilÐ²Ð‚Ñœ (w/o) microemulsion system, and commonly referred to as reverse micelles. W/o microemulsions have tremendous scope for manipulation of reaction conditions to suit the nanoparticle design.
Dynamic light scattering (DLS) theory is a well-established technique for measuring particle size over the size range from a few nanometers to a few microns. A source of light (such as a laser) having known frequency is directed at the moving particles and the light is scattered at a different frequency. This difference in the frequency of scattered light among particles of different sizes is used to determine the sizes of the particles present.
Gold nanoparticles have been used extensively as specific staining agents in biological electron microscopy. According to the Mie Scattering Theory, particle size, shape and agglomeration can cause gold colloids to appear red, violet or blue (Feldheim et al). These nanoparticles of gold have a high backscatter coefficient, so they appear bright in a scanning electron microscope image.
In 1956, the formation of silica particles was observed by reacting TEOS in alkali solution with water in the presence of certain bases (Kolbe). Silica nanoparticles are used to make electronic substrates, thermal insulators and humidity sensors. The quality of some of these products is highly dependent on the size and size distribution of the silica particles (Vacassy).
Fluorescent dyes absorb light at certain wavelengths and in turn emit their fluorescence energy at a higher wavelength. Each dye has a distinct emission spectrum, which can be exploited for multicolor analysis. Fluorenscein-5-isothiocyanate (FITC) has an excitation and emission frequency of approximately 480nm and 520nm respectively. These dyes can be entrapped inside the silica particles (Vanblaaderen) and the spectral characteristics of the dye molecules remains almost intact. Silica encapsulation provides a protective layer around dye molecules, reducing oxygen molecule penetration both in air and in aqueous medium (Santra et al). As a result, photo stability of dye molecules increases substantially in comparison to bare dyes in solution.
Materials and Methods
Triton x100, Cyclohexane, n-hexanol, nanopure water, tetraethylorthosilicate (TEOS), chlorauric acid (HAuCl4), Ammonium hydroxide (NH4OH), Fluorenscein-5-isothiocyanate(FITC), APTS, hydrazine-hydrate, Sodium Citrate (Na3Cit), ascorbic acid, glass vials, stir bars, parafilm, centrifuge tubes.
Preparation of Silica Nanoparticles
To prepare a nonionic microemulsion, 1.77g of Triton x100(nonionic surfactant) and 7.7mL of Cyclohexane (oil) were mixed in a glass vial. Afterwards 1.6mL of n-hexanol (co-surfactant) was added, the emulsion stirred for 5-10 minutes. Four hundred and eighty micro liters (480ÐžÑ˜L) of nanopure water was added to the vial while it stirred. Approximately 5 minutes later, 100 ÐžÑ˜L of NH4OH was added and left to stir, after 10 minutes 50 ÐžÑ˜L of TEOS was added. The vial was wrapped in a layer of parafilm of prevent evaporation and left stirring for 24 hours. Approximately 5-10mL of ethanol was added to the vial at the end of the 24-hour period. The emulsion was left to stand for a few minutes for the silica particles to form and settle to the bottom of the vial. The particles were removed by centrifugation and washed 4 times with ethanol and 3 times with nanopure water.
Preparation of dye-doped Silica Nanoparticles
The dye used to prepare this micro-emulsion was called Fluorenscein-5-isothiocyanate (FITC). The process was carried out in two steps. For the first step, 5 mg of FITC was reacted with 12-14 mg of APTS and approximately 1 mL of ethanol in a clean glass vial. Wrap the vial with aluminum foil to prevent photo bleaching, the solution was stirred for 12 hours. After 12 hours, a microemulsion was prepared by adding 1.77g of Triton x100 to 7.7mL of Cyclohexane and 1.6mL of n-hexanol, a stir bar was placed into the vial and the emulsion stirred for 5-10 minutes. A volume of 480 ÐžÑ˜L of nanopure water was added to the vial while it stirred. Approximately 15 minutes later, 100 ÐžÑ˜L of NH4OH, 50 ÐžÑ˜L of TEOS, and 25 ÐžÑ˜L of the FITC solution were added.
Preparation of Gold Nanoparticles
For this experiment 5% gold nanoparticles was prepared from pure HAuCl4. A 2% solution of hydrazine hydrate was prepared to reduce the gold in HAuCl4. Hydrazine hydrate is an organic compound so it was prepared under the hood. The mixtures were stored at 40 Ð'oC to prevent evaporation. Ten samples were prepared during this experiment; the vials had gold and hydrazine hydrate ranging from 100ÐžÑ˜L to 1000ÐžÑ˜L. The microemulsions were prepared using the steps listed in section I. Successive amounts of solution was prepared by adding 100ÐžÑ˜L of gold to the microemulsion at 10 minute intervals. The same volume of hydrazine hydrate was added to the emulsion at intervals of 15 minutes. Parafilm was placed around the cap of the vials to prevent evaporation and they were left stirring for 24 hours. Afterward, 5-10ÐžÑ˜L of ethanol was added to break the emulsion. The particles were removed by centrifugation and washed 4 times with ethanol and 4 times with nanopure water.
Preparation of 50nm colloid Gold nanoparticles
To prepare these gold particles, 2.54mL of 5mM HAuCl4 was added to 47.5ml Deionized water. The mixture was left to stir and boil. Afterwards, 0.44mL of 1% sodium citrate (Na3Cit) was stirred in quickly. The solution