Ligand Exchange and Aqueous Transfer of Hexanes Soluble Fe2O3(oleic acid)n Nanoparticles to Give Water Soluble Fe2O3(citric acid)n Nanoparticles
So you’ve got nanoparticles – but they’re in hexane. This procedure will get them into water.
- Fe2O3(oleic acid)n nanoparticles in hexanes, ~ 6 mg/mL
- 1.0 M citric acid in anhydrous DMSO
- 100 mM sodium borate buffer, pH 10.0; sodium tetraborate Sigma B3545-500G
- 10 mM sodium borate buffer, pH 8.3
- 20 mL scintillation vial & septum
- 3 x 1” 20 gauge needles
- 5 x Amicon Ultra-15 10K MWCO centrifugal filter, Millipore #
- or Slide-A-Lyzer cassettes
- Vacuum with trap
Important Safety Issues:
- Currently available data suggests that similar Fe3O4 nanoparticles do not have short- or long-term toxic effects on human stem or tumor cells.
- Nonetheless, toxicity of nanoparticles may be highly dependent on many variables, so we take the conservative approach of assuming all nanoparticles are acutely toxic in the short run and chronically toxic in the long run. Since the greatest safety concern is inhalation exposure to inorganic nanoparticles, our standard lab practice is to do all procedures involving pipetting of nanoparticle solutions in an externally vented hood that is approved for nanoparticle work. The biosafety cabinets in 5204 and 5210C are appropriate for this work. Centrifuges that SAA’s for nanoparticles are also located in these locations.
- Heating DMSO to 100C for 3 h appears to lead to some formation of sulfides, which are noxic.
- All materials that potentially come into contact with NP solutions are to be disposed of in nanoparticle containing waste.
- The oleic acid ligands are replaced by citric acid through mass action, or by adding a vast excess of citric acid relative to the existing oleic acid ligands. Therefore it is essential to add a substantial excess of citric acid.
Weigh a scintillation vial w/ its cap. Mass = ______g
Transfer 5 mL ~ 6 mg/mL Fe2O3(oleic acid)n nanoparticles (a transparent, deep brown solution) in hexanes to this pre-weighed 20 mL glass scintillation vial. Dispose of the previous container in nano-contaminated glass waste.
Seal the nanoparticle-containing vial with a septum. Place the sealed vial the hood, and insert a needle connected to a closed N2(g) supply.Pierce the septum with two additional needles as vents. See Fig 1 for a schematic.
Dry under N2 (g) for 0.25-1 h, until the solution dries to a dark reddish brown film. Optional: you may place the vial in a 40C water bath to speed drying.
Apply a vaccuum to finish drying. Remove the septum and replace with its cap
Weigh the capped scintillation vial containing nanoparticles. Mass =_______g; Mass of dried nanoparticles = ________g
Make up a 1.0 M solution of citric acid in DMSO. This corresponds to 0.192 g citric acid /mL DMSO.
Add 0.625 mL 1.0 M citric acid using a micropipettor.(To change scale, use 0.125 mL 1.0M citric acid per mL Fe2O3(oleic acid)n . This is approximately xx fold excess citric acid over oleic acid.)
Add a micro stir bar, and heat the vial for 3 h at 100 °C in an oil bath, to give an opaque, dark reddish brown solution. If necessary, sonicate the vial to promote re-dissolution of nanoparticles on the upper edge of the vial.
Remove the vial from heat and cool to 25 °C. Carefully wipe the hot oil clinging to your vial with a paper towel before touching with gloved hands.
Add the resulting solution slowly to 45 mL of Na2B4O7 buffer, 100 mM, pH 10.0, to produce a cloudy, dark reddish brown solution.
Check the pH of the resulting solution using litmus paper. It should be ~2.
Adjust the pH by slow, dropwise addition of 1.0 M NaOH to 10 (litmus).The solution should now be a clear, dark reddish brown aqueous solution.
Add the solution to a series of Amicon Ultra-15 10K MWCO Centrifugal Filter Devices (max volume ~15 mL) and centrifuge at 3.0k rcf for 15 min at 25 °C, to produce a concentrated solution of water soluble nanoparticles in a final volume of approximately 2 mL. The eluate may form a few soap bubbles on the surface; this results from the displacement of sodium oleate. Dispose of the eluate as sodium borate buffer with a trace of oleic acid.
Transfer nanoparticles to a 1.0 mL 10MWCO Slide-A-Lyzer. Dialyze against 1 L 10 mM sodium tetraborate buffer pH 8.3 at room temperature for 12 h. Replace buffer and re-dialyze for an additional 12 h.
This solution was diluted to 60 mL with Na2B4O7 10 mM, pH 8.3, and divided evenly between 4 Amicon Ultra-15 10K MWCO Centrifugal Filter Devices (Millipore). The samples were centrifuged at 3.0k rcf for 15 min at 25 °C, giving approximately 0.5 mL of a dark reddish brown retentate containing water soluble Fe2O3(citric acid)n nanoparticles, and a clear, colorless eluate. The eluate was discarded.
The particles were diluted to 15 mL with Na2B4O7 10 mM, pH 8.3, and centrifuged at 3.0k rcf for 15 min at 25 °C, giving approximately 0.25 mL of Fe2O3(citric acid)n nanoparticles.
The dilution and centrifugation steps were repeated 5 times, with the final, clear, dark reddish brown solution of water soluble Fe2O3(citric acid)n nanoparticles diluted to a final volume of 4.36 mL in Na2B4O7 10 mM, pH 8.3.
Dynamic Light Scattering (DLS) measurements (Malvern Instruments) performed in buffer at 20 C showed that the particles were narrowly distributed, with a mean diameter of 11 nm.
References & Further Information:
- H. M. Jensen, “Engineering of a synthetic electron conduit in living cells.” PNAS (2010).
- EHS 0344, Safe Handling of Nanoparticulate Matter
- Procedure initially developed by Aaron Albers
- Procedure documented by Caroline Ajo-Franklin