To be specific, I have GNFs (35-40 nm) in HEPES solution and antibodies in PBS? when I mix antibodies (in PBS containing 137 mMol NaCl) and GNFs they tend to flocculate. How to minimize this effect?
A high ionic strength affect the stability offered by hepes to your GNFs. Since they are quite small, few molecules of PEG (or other polimer, I suggest PEG because is know to be quite biofriendly) should stabilize enough your particles in the buffer media.
The problem I face here is, antibodies are in PBS. When antibody is added to GNFs, they immediately flocculate. PEG treatment comes only after gold conjugated antibodies are formed.
The problem here is that the newly formed GNF-antibodies aggregate before PEG treatment.
Your buffer system has too high ionic strength for GNFs. The other possible reason is that the ratio of antibody to per GNF. Enough antibody-modified gold nanomaterials can be stable in PBS for a long time. If the GNFs do not aggragate after mixing GNFs and antibody, you should consider above factor.
Exactly, What I was suggesting is to functionalize the GNFs with few PEG molecule before the transfer in the PBS solution. This should increase the stability without affecting significantly the antibodies adhesion.
As pointed out by Leonardo, ionic strength of the resulting medium is too high. You can do a simple salt titration to estimate the maximum stabilizing concentration. You need to make sure that overall ionic strength of solution in which nanoparticles are kept is always less than MSC. For this you can dilute your antibody in low ionic strength phosphate buffer instead of PBS.
Leonardo's suggestion of increasing nanoparticle stability by surface modification is also good. However, I do feel PEG is not a good choice as you are trying to immobilize proteins on the basis of non-covalent interactions. PEG is known to have non-fouling behavior and is used for increasing the circulation time of nanoparticles in systemic circulation.
Are the GNFs stable if you dialyze them against 500 micromolar citrate? That is the typical starting point, I think, for classic antibody coating of solid gold nanospheres. Coating of solid gold nanospheres would thus be a good practice scenario to work out prior, or in parallel to the more challenging GNF system. Match up the conditions (core concentration, pH, salting steps, buffer type, antibody concentration, time) between spheres and flowers and troubleshoot?
- Badges
- Science topic
- Similar topics
- Chemistry
- Nanotechnology