Hello!

I’m trying to conjugate commercial Gold-PEG-COOH (40 nm) particles to antibodies and have troubles with Gold aggregation (as a black precipitate that cannot be redissolved at vortexing or sonication) at one particular stage. Would be grateful to get any feedback from you what can be wrong.

Below is the protocol I use. It is based on the “Carboxyl Gold Nanoparticles Conjugation Protocol” from CD Bioparticles that I found on the internet. The Gold particle manufacturer hasn’t provided me with the recommended protocol so far (the request was sent):

1. 50 uL Gold-COOH + 50 uL MES 0.03 M + 25 ul EDC in water (at 30 mg/ml) + 25 uL sulfo-NHS in water (at 30 mg/ml).

2. + 1.5 mL of 0.05% Tween-20 in 5 mM Phosphate buffer. Centrifugation at 4200 rpm 10 min rt.

3. Supernatant is discarded, 150 uL ABs (0.5 mg/mL) in 10 mM phosphate buffer pH 7.4 is added. Everything was redissolved well. No black precipitate, only a little bit of pink layer on the tube (that wasn’t redissolved). UV-vis spectra showed only 10% decrease in OD at the peak at this stage (compared to initial Gold-COOH). 2 hrs incubation with ABs.

4. + 1.5 mL of 0.05% Tween-20 in 5 mM Phosphate buffer. Centrifugation at 4200 rpm 10 min rt.

5. Supernatant is discarded, 150 uL Block buffer (1% BSA in 10 mM phosphate buffer pH 7.4). Only part of the solution was redissolved, sonication didnt help. A lot of black visible precipitate. UV-vis spectra shows that only 10% of the gold stayed in the solution.

6. When I tried to precipitate the obtained clear supernatant at centrifugation, the next round of centrifugation resulted again in black precipitate.

The control sample (gold particles gone through the same steps but without EDC/sulfo-NHS and ABs addition) didn’t show ANY precipitate at ANY stage.

Can it be that I have too much EDC/Sulfo-NHS in the solution that destabilize the system somehow?

Plus, would be cool to know if you usually add surfactant during ABs immobilization? Some protocols (NanoComposix) suggest to add PEG 20K during immobilization with ABs.

Thank you very much for any suggestion!!