for coupling via the N-terminus of biochemical species
The NHS-ester reacts immediately with the NH2- terminus of biochemical species to form a covalent bond with the surface (420 kJ/mol). The reaction of carboxyl functionalities with N- Hydroxy succinimide leads to highly reactive esters, which can be easily reacted with nucleophiles e.g. amines, hydrazines. However, due to its high reactivity the NHS ester is susceptible against hydrolysis and is characterized by a relatively short shelf-life. All NHS-activated surfaces should therefore be processed quickly.
There are a number of different approaches to couple on the NHS-surface:
- It is assumed that not all Carboxy groups have reacted to NHS- esters during activation. Thus a negatively charged carboxy surface still remains which in turn supports the physico-chemical adsorption of positively charged probes e.g. NH3+. Hence a protonating media (pH < 5) for the biochemical species getting a positively charge is required.
- A nucleophilic attack on the active ester is also catalyzed under basic conditions (pH > 8,5).
After attachment of the biochemical species the surfaces must be blocked with a blocking buffer containing small molecules that can access all reactive groups within the 3D- Matrix.
PolyAn equips glass slides and polymer slides as well as 96-well plates with 3D-NHS surfaces. Please do not hesitate to contact us, if you would like to functionalize a different format or substrate with our 3D-NHS surface.