about nanoparticles, which have a COOH group on its surface with a tripeptide having a NH2 group, using EDC-NHS chemistry:
can anyone provide information on a detailed procedure or can help me with a reference for it ?
I am confused about the pH to be used during activation and conjugation.
Hi Amit, Please find attached the chapter for carbodiimide coupling from Hermanson. The difference between NHS and sulfo-NHS is their solubility in aqueous solution, sulfo-NHS being more soluble than NHS. You will find these details in the the attached file. I have underlined some important parts on the file.
An article "Y. Zhou, Z. Tang, C. Shi, S. Shi, Z. Qian, S. Zhou. Polyethylenimine functionalized magnetic nanoparticles as a potential non-viral vector for gene delivery. J Mater Sci: Mater Med, 2012 Jul 24." is devoted to this problem. You can find "Synthesis of PEI functionalized magnetic nanoparticles" there (magnetic nanoparticles have COOH on their surface), but you will not have information about pH from the article, unfortunately.
Hi Amit. Activation needs for acidic conditions (e.g. pH 5) and conjugation generally undergoes at higher pH values (e.g. pH 7). Just make sure to choose the suitable buffers for the conditions/solvents you are seeking to apply. Further info: N. Nakajima and Y. Ikada, Bioconjugate Chem (1995) 6, 123-130 -- "Mechanism of Amide Formation by Carbodiimide for Bioconjugation in Aqueous Media"
Amit, in the past I've had good success running both reactions (The activation and conjugation) around pH7. Generally I found a little heat helped speed up the reaction, and I would add both the EDC and the aminated molecule to the particle suspension with moderate stirring at 30-40 C and react over night. For a great overview of many bonconjugation strategies, I would recommend the book "Bioconjugate Techniques" by Greg T. Hermanson
http://www.sciencedirect.com/science/book/9780123705013
I agree with Tom on this, the textbook of Hermanson offers great bioconjugation strategies not only for carbodiimide coupling but many others.
Personally I have performed the activation step in acidic as well as neutral pH conditions, both EDC and sulfo-NHS.
Depending of the stability of your nanoparticles in each pH, you could optimise the reaction conditions to suit your system. Tripeptides are not so bulky as proteins or antibodies and the coupling should go smoothly.
Thank you All for your suggestions. I shall be highly grateful if any of you can send me the e-chapter on conjugation from the textbook of Hermanson, as it is not subscribed by our institute.
I would like to ask Cristina, that, I have NHS instead of sulfo-NHS, I am afraid that does it going to make a big difference.
Thank You.
Thanks Ian. My nanoparticles are stable in most of the solvents including water and buffer. According to your suggestion I should simply mix all the components and keep it for stirring for overnight. Dont I need to add NHS. It would be very kind of you, if you can message me the detailed experimental procedure for coupling. Thanks Again.
Hi Amit, Please find attached the chapter for carbodiimide coupling from Hermanson. The difference between NHS and sulfo-NHS is their solubility in aqueous solution, sulfo-NHS being more soluble than NHS. You will find these details in the the attached file. I have underlined some important parts on the file.
Thanks a lot Cristina!!! The attachment is really helpful to me. I got almost all the answers there. I am obliged to you for taking the pain of marking the important parts for me.
Can anybody tell how to determine the conjugation done or not using EDC and NHS. Of course FTIR gives the functional group. But i wanna know is there any direct evidence to prove crosslinking?
Check out the book "Nanomaterials for Medical Applications" from Elsevier. It has a collection of procedures for various researchers on how ton conjugate to different nanomaterials.
http://www.amazon.com/Nanomaterials-Medical-Applications-Zoraida-Aguilar/dp/0123850894
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