MicroSurfaces, Inc. enables the proteomics & biosensor industries
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Advanced Surface Coatings for Protein Microarrays, Microfluidics & Biomedical Research


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Application note:

The zero-background NHS surface for protein, peptide, & antibody immobilization

NHS surface
Figure 1. The zero background NHS surface consists of the efficient leaving group, NHS, tethered to the high-density PEG coating. A protein molecule is attached via –NH2 group(s) but is otherwise repelled from the PEG coating. Excess NHS groups on the surface are easily de-activated in a simple washing step. The same immobilization reaction applies to peptides, antibodies, and oligonucleotides..
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.
Figure 2. Fluorescence images of peptide microarrays fabricated on NHS/PEG/glass slides and detected by immunostaining. The diameter of each spot is ~100 um.

While the NHS/PEG coated glass slides are ideal for protein (See Technology), 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, cover slips, 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.

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