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 • • • • • 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