I diluted EDC/NHS and aptamer in water. The aptamer attached to the surface 2 mounts ago but now it did not attach.
EDC is hygroscopic and thus it starts to hydrolyze. Was the original EDC new? Are you using the same reagent?
It is suggested to check the activity of EDC, which may suffer hydrolysis. For example, instead of using expensive aptamers, just use the small molecules with primary amine (i.e., some amino acids), which should be easily characterized. Hope this helps.
I do two step process for surfaces. 20 mg/mL EDC, 2 mg/mL NHS in 100 mM MES buffer (pH 4.8) for 25 min (RT), then NH2 group for 2 h RT or 4 C overnight.
Store aliquots of EDC and NHS under dry nitrogen at -30 C. These are very susceptible to hydrolysis.
You should not see any clumping of the reagent. And EDC will begin to smell "fishy" as it hydrolyzes.
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