I have functionalized 20 nm AuNPs with SH-PEG5k-COOH and SH-mPEG5k in a 9 :1 ratio. The final solution is around ~1 OD in DI water. To react with EDC/Sulfo NHS, I am using the following conditions.
MES buffer at pH 5.4, 50 mM, 5 mL
AuNP@PEG-COOH/mPEG OD ~1, 1mL in MES buffer
EDC/NHS ~ 10 ug each (stock solutions are prepared in DI water), 100 uLwas added soon after preparing
Reaction time- tried both 15 min, 30 min
Issues with aggregation/stickiness after addition of EDC to carboxylated particles are extremely common.
A big factor is usually the charge at the surface. The carboxyl groups enhance colloidal stability by a repulsive effect but in activating with EDC/NHS you eliminate the charge and generate neutral highly reactive species.
The detail missing from your description is why you are activating the surface. I assume that a protein is added next? You may wish to consider coincubation of particles and protein and NHS/EDC. This requires careful optimization of ratios to avoid widespread crosslinking but it has the advantage that the conjugated protein can enhance particle stability.
Another thing to consider is the PEG mix - do you have enough/too many carboxyls, which is easily explored by simply varying the ratio of the two thiolated molecules. For example, more carboxyls but with a submaximal EDC/NHS activation may improve colloidal stability and yet still allow conjugation of your protein of interest.
Nick Gee Thank you very much.
Yes, I am adding protein as the next step. I will try the coincubation. Do you suggest me to do it in PBS buffer instead of MES?
I am adding more SH-PEG-COOH than SH-mPEG (9:1 ratio). Are you suggesting increasing the SH-PEG-COOH amount more than this ratio?
Sorry I read 9:1 the wrong way round. I suspect the edc/nhs ratio and the individual concentrations are what you’ll need to work on to improve things. You can’t increase carboxyls much more!
You could use PBS but it interferes with edc reactions and will reduce your rate of reaction. If you try varying the EDC you can change the rates and maintain your current buffer conditions if you prefer.
The literature shows a huge variation in conditions used for conjugation, anything from 0.1mM up to 100mM EDC, and with other components to vary too there’s a bit of optimisation work to do here. Gut feel is that you’ll need to be towards the bottom end of the range, but just follow your data.
I have noticed the same when carrying out EDC/NHS linkage of proteins to gold particles in contrast to using passive adsorption. Solution: make the reaction glass vial and stir bar surface hydrophobic by simple treatment with Sigmacote (Sigma/Aldrich)