Calcium Phosphate Nanoparticles Synthesis
Procedure:
• Make sure the hood is clean and free of all chemicals other than the ones required.
This will minimize contamination.
• Use Millipore water (001D) for synthesis as well as purification. Collect 4 L of
Millipore water in a 4 L solvent bottle. Make sure that the resistance of Millipore
water is between 12-14 (will be shown on the display).
• Degas the water in Dr. Vigh’s lab (room 218) on an aspirator for 2 h to remove
carbondioxide.
• Check the pH of the water and adjust it to 7 with 0.1 M KOH.
• In the mean time degas 200% proof EtOH (available from stock room) in our lab
(301B) for 2 h.
• The degassed water and EtOH will be good enough for at least a week.
• You may want to setup multiple reactions at the same time to avoid degassing
your solvents every now and then.
• Prepare 10-2 M calcium chloride solution in the above degassed water (make sure
to sonicate for 10 min to dissolve calcium chloride completely).
• Likewise prepare 6 x 10-3 M disodium phosphate solution from the above
degassed water.
• Disodium phosphate is less soluble in water than calcium chloride; please stir the
solution for 30 min. for complete dissolution.
• Prepare disodium silicate 8.3 x 10-4 M solution in water. Sigma Aldrich sells
disodium silicate as solid as well as solution. Make sure you buy solution and
dilute according to your need. Solid disodium silicate is almost insoluble in water.
• Prepare 10-3 M sodium citrate solution in degasses water. Sodium citrate is
available as a solid and is easily dissolved in water by sonication for 10 min.
• The dye to be encapsulated can be dissolved in EtOH/water/THF. Most organic
fluorophores are dissolved in EtOH or water. Conjugated organic polymers are
dissolved in THF due to solubility issues. The concentration of organic
fluorophore should be 10-3 M. Concentration of conjugated organic polymer can
be 10-4 or 10-5.
• Make 29 % Igepal CO-520 (Sigma Aldrich) solution in cyclohexane (stock room).
Do not handle with bare hands; Igepal CO-520 is a cancer suspecting agent.
• Prepare microemulsion A: Add 650 µL of 10-2 M calcium chloride solution in
water to 14 mL of 29 % Igepal CO-520 in cyclohexane and stir under nitrogen for
1h
• Prepare microemulsion B: Add 650 µL of 6 x 10-3 M disodium phosphate
solution, 65 µL of 8.3 x 10-4 M disodium silicate solution, 1mL of 10-3 M dye
solution in EtOH or water to 14 mL of 29 % Igepal CO-520 in cyclohexane and
stir under nitrogen for 1 h.
• Add Microemulsion A to microemulsion B using a syringe pump. Addition
should be slow (~30 min) to avoid precipitation of large particles. Make sure the
solution is under constant stirring. On a digital display stirring plate adjust stirring
to 500 and on a non digital one adjust the knob to 5. Check stirring every half
hour or so to make sure stirring is on. The digital display stirring plates sometimes
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stops for no reason. The solution has to be stirred for 6 h for conjugated polymers
and 24 h for organic fluorophores.
The reaction is quenched by adding 225 µL of 10-3 M sodium citrate and stirring
for 30 more minutes.
The reaction mixture is now diluted by adding 50 mL degassed EtOH to break the
micelles and loaded on to a normal silica column. Purified by initially eluting with
EtOH to remove the non encapsulated dye and the surfactant. Increase polarity of
solvent to 30/70 water/EtOH to elute the nanoparticle. You can also use 40/60 or
50/50 water/EtOH for elution.
Make sure the nanoparticles are collected and stored properly in a fridge or away
from direct light. This will increase its bench life.
Please take an AFM to makes sure you have the nanoparticles.
A portion of the above purified nanoparticles can be further purified by passing
through 15 µm (stellar Phase Inc. PA) silica bead column and eluting with water
for further purification. This step is usually time consuming (anywhere between
4-5 h due to small size of silica and make sure to use a short column). 15 µm
silica bead is very expensive, please do not throw them away, wash and reuse it.
microemulsion B
dye cargo
microemulsion A
Na2HPO4
CaCl2
Na2SiO3
Igepal CO-520 in
cyclohexane
25 oC
6 h or 24 h
Igepal CO-520 in
cyclohexane
calcium phosphate
particle formation
6 h for conjugated organic polymers
24 h for organic fluorophores
sodium citrate, 25 oC, 30 min.
quench the reaction
purified via vdW-HPLC
calcium phosphate
nanoparticles with
carboxyl group on surface
Sodium silicate Na2SiO3 is for nucleation of nanoparticles.
O
5
OH
C9H19
Igepal CO-520 for micelle formation
+Na–O C
2
CO2H
CO2–Na+
OH
sodium citrate
Thanks
Jiney