I am trying to anchor aptamer on graphene surface. For that, I am using PBASE-linker to anchor amino-linked DNA. But it seems that the aptamers are not anchored. I have two concerns:
1. As like TCEP to break the disulfide bonds between two thiolated DNAs, is their any similar treatment required to break the amine bonding between two amino-linked DNAs (if any)?
2. Is their any suitable pH level that enhances the NHS crosslinking?
thanks in advance.
Hi Niazul, N-succinimidyl esters are notoriously unstable at physiological pH.
I have 2 suggestions. 1) incubate the aptamer with PBASE first (in organic solvent if you can) before adding it to the graphene.
2) use partially oxidized graphene with terminal -COOH groups. Then you can do a simple carbodiimide (EDC) coupling.
Thank you a lot Steingrimur. I would appreciate if you could provide me with some good references with protocols for number 1 suggestion.
Use DMF as reported by
Article Scalable Production of High Sensitivity, Label-Free DNA Bios...
Thank you. We also use DMF as a solvent for PBASE. Then incubate in DNA in Phosphate buffer saline which has a pH of around 7.0. I am wondering if this pH level has any harmful effect on the NHS linking between PBASE and amino-linked DNA.
Thanks again.
NHS esters are always susceptible to water hydrolysis. This reaction is favored at high pH. You need to add sufficient amount of aptamer to compete with this reaction.
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