In the biomedical applications of photothermal therapy, tumor imaging and metal-enhanced fluorescence, gold nanoparticles have become increasingly popular in the past ten years.
For these applications, high-quality gold nanoparticles with monodisperse sizes and aspect ratios are required. In this article, a simple density gradient technique is demonstrated that uses a high-speed Avanti JXN centrifuge for the purification of monodisperse gold nanorods from a polydisperse sample.
In the field of biomedical imaging, gold nanorods hold great promise. They have very strong absorption peaks in the near-infrared and visible region due to a plasmonic effect; the peak wavelength is directly determined by the aspect ratio of the gold nanorod. It is important in biological imaging to have optically pure gold nanorod samples, which also need physical purity. The gold nanorod synthesis process usually causes a degree of impurity in the form of gold nanospheres which do not elongate and non-optimal gold nanorods that have slightly different aspect ratios. As gold nanorods and gold nanospheres (AuNS) are made up of the same constituent element, have the same surface coating from synthesis and are also of similar sizes, their separation can become a problem.
Density Gradient Centrifugation (DGC) is very effective for separating nanoparticles of similar sizes but different densities due to the very small shifts in volume/surface area ratios. In this article two pure gold nanorod samples were used, one 10nm x 41nm with 800nm plasmon (4.1 aspect ratio) and the other 25nm x 60nm with 650nm plasmon (2.4 aspect ratio). The samples were mixed and then separation was performed with a single-step DGC on the new Avanti JXN instrument using a JS-24.15 rotor. Based on optical spectroscopy, the isolated gold nanorod samples showed the same purity as the original samples did before they were mixed.
Density Gradient Centrifugation of Gold Nanoparticles
Gold nanorods of10nm diameter (808nm plasmon peak) and 25nm diameter (650nm plasmon) were concentrated to 0.05 mL, by pelleting 3mL of each in the Beckman Coulter Microfuge 16 at 10,000 x g for 5 minutes, followed by their resuspension in water with 0.01 CTAB. The density gradient was set up manually in 15mL polyallomer centrifuge tubes (P/N 361707), as shown in Figure 1.
Figure 1a. Absorption spectroscopy of the samples of gold nanorods before mixing them together.
Figure 1b. Absorption spectroscopy of the pure samples of gold nanorods after mixing them together before Density Gradient Centrifugation.
In a Branson MI8 sonicator, sonication of both gold nanorod samples was carried out for 5mins. The samples were then mixed and layered on top of the density gradient. Next, they were centrifuged at 10,750 x g at 25° C for 15 minutes using a JS-24.15 rotor in the Beckman Coulter Avanti JXN-30. The deceleration and acceleration rates were fixed at 3. Fractions with a 300µl fraction volume each were collected following the run. Using Paradigm, these fractions were scanned for peaks and then pooled based on the 808nm and 650nm peaks.
Buffer exchange was carried out by pelleting the pooled fractions using Microfuge 16 and resuspending in 0.0IM CTAB. This was done three times and 250µl of 0.0IM CTAB was used for final suspension of the pellet. Using a DU800, spectrophotometer readings of the collected peaks and the mixed sample were taken prior to centrifugation to observe the separation.
Conclusion and Discussion
After analysis of the pure samples by absorption spectroscopy (Figure 1a), the samples were mixed together and the absorption spectrum (Figure 1b) was reanalyzed. It was possible to test how pure the gold nanorod samples were by analyzing the longitudinal plasmon peak absorption (800nm and 650nm in this case), with the transverse plasmon peak absorption (515nm for both samples).
In the case of the pure samples, the 650nm plasmon gold nanorods had an absorption ratio of 2.32 when comparing 650nm with 515nm. For the 800nm plasmon gold nanorods, the ratio of 800nm to 515nm was 3.85. After the centrifuged mixture was fractionated and optically pure samples were collected, the absorption spectrum (Figure 2) was analyzed again.
The 650nm/515nm ratio was found to be 1.91, which was almost as high as the pure 650nm plasmon sample. Interestingly, the 800 nm/515 nm ratio was 4.54 for the 800nm Plasmon sample, which was even higher than the pure sample.
This shows that some AuNS contamination was present in the original, pure 800nm plasmon sample that was separated out by the DGC run.
Figure 2. Absorption spectroscopy of separated samples of gold nanorods after Density Gradient Centrifugation.
Figure 3. Images of centrifuge tubes with gold nanorods: (a) before Density Gradient Centrifugation; and (b) after Density Gradient Centrifugation.
Figure 4. Flow Chart.
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About Beckman Coulter
Beckman Coulter develops, manufactures and markets products that simplify, automate and innovate complex biomedical tests. More than a quarter of a million Beckman Coulter instruments operate in laboratories around the world, supplying critical information for improving patient health and reducing the cost of care.
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