Hi Joseph, the problem with using EDC/NHS coupling is that the NHS ester is very unstable and I think it is counterproductive to use an NHS ester for simple coupling. I have no idea why researchers are still using it.

Most coupling of -NH2 and -COOH can be done with EDC alone with no NHS ester intermediate.

The best way to functionalize Au particles is to use a bifunctional reagent with a free -SH and a -NH2 or -COOH (glutathione comes to mind). Then you can do straight coupling using only EDC in MES buffer, pH ~5.

what do you have on the surface of the nanoparticles? If you are experiencing stability issues use SH-peg-amine first.

Thanks Steingrimur Stefansson and Omar, but the AuNPs is capped with Glutathione which my Supervisor wants me to use.

Stefansson can you please get a procedure for that because i tried coupling with EDC at pH of 6 but still didnt work. I dont know if my approach is wrong .

Thanks.

Could you please provide details on the conjugation. Concentrations and conditions please? Do you see precipitation by only adding edc?

Okay Omar. the molar ratio of EDC: Rhodamine is 10:1, EDC:NHS is 1:1 and the AuNP:Rhodamine is 1:2 respectively. I usually weigh 0.0065g of RhodamineB, 0.0261g of EDC and 0.0295g of Sulfo NHS all dissolved separately in 4ml of MES buffer at pH 6.0. After that i pick aliquot of each stock solution and i add EDC first to the RhodamineB before adding the NHS and leave to react at room temperature for 15mins. After 15mins i add the AuNPs and react the whole mixture for 2hours before testing under gel electrophoresis.

I see precipitation after i add Sulfo NHS.

Thank You

According to what you describe you see some precipitation before adding the nanoparticles. Correct? How is the aunp/rhodamine ratio calculated?

Yeah i see some precipitation before NPs is added. The ratio of AuNP/ Rhodamine was calculated by using the mole of GSH since thats what will bind with the rhodamine. So GSH mole was gotten from Gold to GSH ratio which is 22:10.

Get rid of sulfo-nhs and see if precipitation does not occur or reduce the amount of both edc and sulfo nhs. You need to fix this precipitation issue first before adding nps.

Thanks Omar. I will get rid of the sulfo nhs and try coupling at pH of 5. Also i will try reducing the edc and the sulfo nhs from 10:1 as compared to the Rhodamine B to 5:1 ratio.

Please can i know which temperature condition is best for EDC reaction? I usually use 25 degrees celsius.

Since you are not using proteins or anything like that RT is fine

Okay. Thank You.

Hello. I performed the experiment with reduced amount of EDC dissolved in MES of pH 5. After testing with gel electrophoresis, the AuNPs got separated from the RhodamineB but looks a bit darker.

What might be the cause?

The gel analysis happened after remotion of free rhodamine? What kind of electrophoresis are we talking about?

I used Agarose Gel Electrophoresis. I did the electrophoresis after 2hours of reaction between EDC and Rhodamine B with AuNPs and also tried after 24hours but same results.

Did you add beta mercaptoethanol or dithiothreitol to the sample buffer? How did you remove free rhodamine after reaction? If you are able to separate nps by centrifugation, shine uv light to the pellet and you should see fluorescence. Also after washing the nps a UV-Vis analysis should reveal changes if functionalization occurred. A fluorimeter should also give a reading when compared to a control. All of these can be performed only after washing of the nps.

I didnt add mercaptoethanol. So please after centrifugation is it necessary to add MES buffer to dissolve the precipitate so you can run electrophoresis? Or electrophoresis is no more important as you didnt mention?

Hi Joseph, this will be tricky since glutathione has 2 -COOH groups and 1 -NH2 group and rhodamine has 1 -COOH group.

Any EDC/NHS coupling will give you multiple products and probably cause NP cross linking and precipitation.

If it were me, I would abandon the glutathione coating and try something like 3-Aminopropane-1-thiol hydrochloride hydrate

https://www.sigmaaldrich.com/catalog/product/aldrich/otv000791?lang=en®ion=US

It has one thiol that binds Au and one amine group that would be easy to couple to rhodamine B, since it has one -COOH group.

Thanks Stefansson, but what if 2 mercaptoethanol is added to deactivate EDC so it doesnt react with the 2-COOH of glutathione?

Any idea on the charge of the final conjugation of rhodamineB with AuNPs capped with glutathione using EDC reaction.

Do not add mercaptoethanol

We have used EDC. The most critical steps is when you add EDC to the reation, should be quickly. Also the coating of AuNp with cooh should be checked.

Use INFRARED spetra to confirm

Regards

Thanks Omar and Antonio.

Antonio please which pH of MES did you use? INFRARED spectra to confirm the coating of AuNPs with GSH?

I have attached a photo of electrophoresis under UV light for rhodamineB activated with EDC using MES pH 4.0 and conjugating with AuNPs capped with GSH. Please what do you think about the results?

Please describe lanes

In addition to Omar's suggestion, run unconjugated NPs so you can see if you have multimers due to the conjugation.

Okay Omar. From the side that shows moving bands. The first is at pH 4 without NHS, the second is at pH 4 with NHS, the 3rd which moved in the opposite direction is free AuNPs and the 4th is at pH 5 without NHS and the 5th one is at pH 5 with NHS.

Thanks Stefansson, but unconjugated AuNPs is what is found at position 3. it moved towards the positive charge.

o Please what can be done to improve the conjugation yield when using EDC/NHS

Convince your supervisor that coating the Au NP with glutathione is a bad idea. Use cysteamine instead

https://www.sigmaaldrich.com/catalog/product/aldrich/m9768?lang=en®ion=US

Now you have AuNPs with primary amines and rhodamine with free carboxyl groups. This coupling should be easy with EDC alone.

Forget about making an NHS intermediary. It is an extra unnecessary step if you want to couple amines to -COOH.

Okay Stefansson. Thanks

You must be cautious when using cysteamine, it can make your particles disappear.

Please how Omar? I dont really understand.

Yeah Omar, I don't get it either.