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-hydroxysuccinimide leads to highly reactive esters, which can be easily reacted with nucleophiles e.g. amines or hydrazines. However, due to its high reactivity the NHS-ester is susceptible to 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 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, coverslips 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.