Standard Operating Procedure Preparation of Agar Phantom for MRI Calibration

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Last Edited: HLR, 9/25/14
Purpose:
To prepare nanoparticles of different concentrations in NMR tubes in 1% agar.
Materials:
NMR tubes
NMR tube caps
Agar
50mL Beaker
MQ Water
3/8 stir bar
hotplate w/ magnetic stirrer
watchglass

4mL glass vials

Transfer pipets
timer
vortexer
falcon tube (50mL, PET)
Teflon holders (homemade)
Saline solution (0.9% NaCl(aq))

Methods:
Tube Preparation:
1. Take NMR tubes & use a sharpie to mark ~ 1.5 2 from the bottom of the
tube.
2. Use a file to score the outside of the tube at the sharpie line.
3. Carefully, break the tube.
WARNING: Use cut resistant gloves when breaking glass.
WARNING: Wipe up any small pieces of glass remaining on the lab
bench with a wet paper towel.
4. The entire NMR tube can be cut into 5-6 pieces.
The pieces that are open on both ends can be sent to the Chemistry
glass shop to have a single end closed off. This will cost ~ $50/80 cut
tubes, but may also take 2 -3 months to get through the queue.
Prepare Agar:
1. Prepare a 2% agar solution, the procedure below is for making 10mL of agar.
You will need at least 0.5mL of agar for each NMR tube.
2. Measure 0.2g agar into a 50mL beaker.
3. Add 10 mL of MQ-H2O.
4. Add a 3/8 stir bar to the solution.
5. Set beaker on hot plate.
6. Set stir setting to maximum (1200 rpm).
The agar should mix with the solution, resulting in a cloudy solution.
7. Set hot plate to above 200F.
8. Cover the beaker with a watchglass.
9. Once agar is fully in solution (solution become transparent), turn the hot plate
down to 90F.
Avoid allowing the agar to boil, as this can increase the agar
concentration.
Prepare the Samples:
1. Put 0.5 mL of sample into a 4mL glass vial. Ensure that a transfer pipet can
easily fit into the vial.
2. All samples should be prepared at 2x the desired concentration of the
phantom.
3. Mark the caps of all the NMR tubes for your samples.
Remember to also create a control with 0.5 mL of the matrix the
nanoparticles are suspended in (H20, PBS, cells, etc.).
Each phantom for the 9.4T-31cm magnet can hold up to 6 NMR tubes.
Recommended final concentrations for NMR tubes would be: Blank,
0.01, 0.05, 0.1, 0.5, 1.0 mg Fe/mL.
Prepare the NMR Tubes:
CAUTION: Have everything ready to use before starting this process.
1. Pipet 0.5mL of hot agar into 4mL glass vial with sample.
2. Cap 4mL glass vial.
3. Vortex sample and agar for 30s.

NOTE: optimize time as necessary to have a homogeneous mixture

without the agar setting outside of the NMR tube.
4. Use a transfer pipet to move sample from the 4mL vial to the NMR tube. It is
important to avoid any bubbles in the NMR tube.
5. Allow samples to sit at 4C overnight to fully set.
Troubleshooting Recommendations:
Make sure the agar is hot, this will prevent it from setting early.
While putting the transfer pipet into the NMR tube, slowly fill the tube
with the agar sample.
If you get an air bubble at the bottom of the tube, stop filling the tube.
Try to flick the tube or use a strong flicking motion with your write to
create a downward jolt to move the sample to the bottom of the tube.
Prepare the Phantom:
1. Insert tubes into the phantom holders.
2. Separate the holders to hold the NMR tubes straight.
The area between the Teflon holders should indicate the best location
to image the NMR tubes.
3. If the NMR tubes do not stay straight, it may be required to cut the edges of
the top of the NMR cap of the NMR tube in the center.
4. Note the location of the markers (big wedge, little wedge, double wedge) in
relationship to your sample locations.
5. Put the tubes in the Teflon holder into the 50mL falcon tube.
6. Fill the falcon tube with the samples all the way to the top with saline.
7. Fill the falcon tube cap with saline solution and quickly cap the falcon tube.
Avoid introducing any bubbles into the falcon tube.