Hello, I have a trouble in EDC/NHS reaction.

I functionalized NH2 group on hydroxyapatite with alendronic acid, and confirmed that through XPS analysis.

As a next step, I want to coat with carboxylic acid terminated Poly(N-isopropylacrylamide) (PNIPAM-COOH, MW 5000) through EDC/NHS reaction, but the reaction did not proceed.

Furthermore, zeta-potential value increased from +15 to +26. (I expected the value should have decreased due to the PNIPAM-COOH)

My process is as below:

1. Mix PNIPAM-COOH in MES buffer (pH 5.05) with EDC solution (MES buffer) and stirring 30 mins.

2. Add NHS solution (MES buffer) and stirring 1 h mins.

3. Add hydroxyapatie-NH2 (HA-NH2) and stirring overnight (~12 h).

4. Washing with DW

I have agonized for many weeks, but cannot find the answer. Please help me.