NHS surfaces

Application Note:

The advantage of this surface is that it allows for very fast immobilization and the remaining chemical groups are easily washed off to expose the zero background PEG brush. Ideal for ultra-small quantities and crude tissue samples.

In many proteomics applications, one is interested in the facile and covalent immobilization of protein molecules without the use of any special tag or chemical modification. This is most conveniently achieved via chemical reactivity towards the commonly available –NH2 groups on the surface of protein molecules. One of the most efficient leaving groups towards –NH2 is N-hydroxysuccinimide (NHS) attached via an ester bond. We have developed an NHS surface based on the zero background PEG coating, as shown in Figure 1. It allows for fast immobilization reactions with the remaining NHS groups easily washed off to expose the zero background PEG coating. In subsequent assays, the PEG functionality ensures that binding of particular molecules to the surface is only through the specific interaction with the immobilized protein molecule and the commonly seen background problem is solved without the need of a blocking step. To demonstrate the performance, we show in Figure 2 peptide and protein microarrays fabricated on NHS/PEG/glass slides. Briefly, nanoliter droplets of peptide (21 amino-acids) or protein (fibrinogen) solution containing 10% glycerol are deposited on the glass slide with a robotic arrayer and incubated for 10 minutes. NHS-groups in remaining area are removed by a deactivating buffer for 30 minutes at room temperature. The immobilized peptide or protein on the surface is detected by incubation with the primary antibody specifically against the peptide or fibrinogen, followed by wash and incubation with cy3-conjugated secondary antibody. The glass slides are imaged on a laser scanner. The most important result is the exceptionally low background due to the PEG coating.


Fig. 1. ZeroBkg® NHS activated surfaces for the immobilization of proteins, peptides, & antibodies	Fig. 2. Fluorescence images of peptide (left) and protein (Fibrinogen, right) microarrays fabricated on NHS/PEG/glass slides and detected by immunostaining. The diameter of each spot is ~100 μm.

While the NHS/PEG coated glass slides are ideal for protein, peptide, and antibody arrays, they are also useful as low background surfaces for other microarrays, such as oligonucleotides, carbohydrates, and other small molecules. The non-fouling property of the high density PEG coating becomes critically important when one uses such an array for the study of complex biological samples, such as plasma or serum. In order to detect molecules of low abundance, such as cancer biomarkers, one needs to minimize non-specific adsorption of other abundant biomolecules.

These coatings are available on standard microscope slides, coverslips, silicon wafers. We also provide customer coating service for specific customer samples. Our customers have successfully applied the NHS/PEG surfaces for a range of applications, including protein sensors, protein microarrays, single molecule spectroscopy, biological atomic force microscopy and other biophysical studies.