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Autor: anton 08 March 2011
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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’Â°C 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 was left boiling and stirring for approximately 10 minutes after adding Na3Cit. This method did not require washing with ethanol; the samples were washed three times with nanopure water.
Preparation of 100nm colloid Gold Nanopaticles in Water
For this experiment, a 50mg/L seed was required. The method for preparing 50nm colloid gold particles was implemented in preparing the seed, the only difference being that 1.5mL of Na3Cit was added to the solution instead of 0.44ml.
5mL of 5mM HAuCl4 was mixed with 0.25ml of 50mg/L seed and 150mL of nanopure water. The solution was left to stir for 20 minutes, and then 100mL of 0.44mM ascorbic acid was added drop- wise slowly to the solution. Washing the solution was unnecessary because it was prepared in water.
After the ethanol is added, pour the contents of the vial into centrifuge tubes and balanced. The vials were placed into the Beckman JA-21 centrifuge. The speed of the centrifuge was set to 9000rpmÐ²Ð‚â„¢s. The centrifuge ran for 20 minutes then the supernatant liquid was removed from the tubes until about 2mL were left trying not to disturb the particles. Fresh ethanol was placed into the tubes to about 20mL, it was placed on the vortex for approximately 5 minutes then sonicated on maximum making sure there are no clumps left and nothing is settling down at the bottom. The tubes were balanced again and the steps above were repeated 3 times with ethanol and 4 times with nanopure water.
Sizing the particles
The particles in microemulsion were sized using Dynamic Light Scattering. The instrument used to size the particles from the emulsion after washing is called the microtrac nanotrac. Before sizing the samples, the laser was washed with nanopure water and placed in flask containing deionized water. This will be the Ð²Ð‚?zeroÐ²Ð‚â„¢ level for the sizing. Each sample is placed in a clean vial and the laser is placed into the vial. This machine is modified to measure gold particles. The machine was set to run 3 times with each run time being 90 seconds. After three runs, the machine reports the average particle size and volume; the standard deviation is also recorded and reported.
The CPS Disc Centrifuge was utilized to size the colloid gold nanoparticles. This instrument uses density gradient centrifugation and Stokes settling equations to size particles from 40 microns to 10 nanometers. A small sample is placed in the centrifuge and it reports the mean size and weight of the particles, the PDI is also reported and recorded.
Results and Discussion
Table 1: Data recorded from the micortrac nanotrac for silica nanoparticles in microemulsion
Sample # Volume of Water (Ð¿ÐƒÂL) Mean Number (nm) Standard Deviation
1 380 161.8 225
2 480 114.8 106
3 580 95.3 79.7
4 680 105.7 119.2
5 780 115 118.1
6 1000 121.5 245.8
The results were shown as a plot of volume of water vs. size of particles in figure1. The emulsion with 380Ð¿ÐƒÂL of nanopure water had the largest particle size of 161.8 nm, while the particles in the emulsion with 580Ð¿ÐƒÂL were the smallest at 95.3nm. The results are shown as a plot of volume of water versus particle size (fig1). It was observed that as the volume of nanopure water in the silica micro emulsion increased the particle size decreased. The fact that the data showed larger particle sizes in the emulsion with the smallest volume of water was expected, because with that amount of water in the vial the sizes should be large. It was not expected that the smallest size be not reported in the micro emulsion with the largest volume of water. This could mean that the larger particles had settled at the bottom of the vial and manipulated the results.
Table 2: Data collected from the microtrac nanotrac for gold nanoparticles in microemulsion
Volume of Gold (Ð¿ÐƒÂL) Mean Number (nm) Standard Deviation (nm)
100 74.5 89.8
200 57.3 54.9
300 58.3 51.6
400 33.2 48.2
500 55.3 99.6
600 36.1 50.7
700 41.6 61.1
800 33.3 47.6
900 48.4 49.5
1000 64.3 117
The largest particles were in the vial with 100Ð¿ÐƒÂL of 5% gold, this was reported as 74.5 nm by the nanotrac and the smallest reported as 33.2 nm, were in the vial with 400Ð¿ÐƒÂL of 5% gold solution. The trend of the data recorded was observed to be that the particle size was decreasing, as the volume of gold was increasing (fig2). As noted before, it was odd that the smallest size reported were not in the micro emulsion with 1000Ð¿ÐƒÂL of gold. This could be because the particles were not sonicated properly and the larger particles were allowed to settle. When this happens, the laser from the nanotrac gives results only from the particles it is able to pass through which would be the smaller particles that have not settled to the bottom of the vial.
Table 3: Data recorded from the CPS for colloid gold in water
Sample # Total vol (mL) Au conc (mM) Au Vol (mL) Seed Vol (mL) Acid Conc (mM) Mean Number (nm) Mean Weight (nm) PDI (DW/DN)
1 36.025 5 1 0.025 0.44 65.8 66.3 1.0086
2 36.525 5 1.5 0.025 0.44 74 78.7 1.0637
3 37.025 5 2 0.025 0.44 82.7 82.5 1.0562
4 37.525 5 2.5 0.025 0.44 106.8 110.9 1.0377
5 38.025 5 3 0.025 0.44 195.1 181.6 1.7409
6 36.025 5 1 0.025 0.44 76.6 80.2 1.0467
7 36.525 5 1.5 0.025 0.44 78.5 85.2 1.0985
8 37.025 5 2 0.025 0.44 102.7 157.8 1.1723
9 37.525 5 2.5 0.025 0.44 113.6 216.2 1.1322
10 38.025 5 3 0.025 0.44 202.5 218.2 1.0775
The results for the colloid gold syntheses were shown using a plot of volume of gold present vs. particle size. The results show that it is possible to use the seed mediated method to grow bigger particles from smaller ones. The procedure was repeated and the data showed that there is a minimal error of Ð¿â€šÂ± 20nm size difference for the particles (fig 3). It is evident that the volume of gold used plays an important role in the size of particles. The data showed that as the volume of gold increases the size of particles will also increase (fig3).
The synthesis of gold and silica nanoparticles was accomplished. The particles were successfully characterized using Dynamic Light Scattering.
The goal for next semester is to duplicate the results as a single process. The data was obtained in stages, it is hoped that the experiment can be done in less time and the results will be the same. I intended to examine the samples using the Scanning Electron Microscope (SEM). The gold particle sizes will also be measured using the UV/VIS spectrophotometer, in hopes of producing the same results.
Thus far I have been able to prepare 200nm particles using the seed mediated method. I intend to continue using this method to prepare bigger particles using the 200nm solution as the seed.
This work was supported by the Particle Research Center (PERC) at the University of Florida. The authors acknowledge the influence and direction of Dr. Brij Moudgil, director of the Particle Research Center. Any opinions, findings and conclusions expressed in this material are those of the author(s) and do not reflect those of the PERC.
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