Maleimide surfaces

Application Note: 
Covalent linking between a maleimide group and a sulfhydryl group is one of the most selective, facile, and convenient reactions in bioconjugation chemistry. We have now made this favorite reaction available on the ZeroBkg® surface.

Development of a solid-phase environment that provides optimum bioactivity without biomolecule loss, displacement, or surface migration is a common goal of research scientists, clinical laboratories, and diagnostic kit manufacturers. One likely approach is the facile and covalent immobilization of protein molecules without the use of any special tag or chemical modification. This can be achieved conveniently via chemical reactive group, maleimide, towards the available sulfhydryl (–SH) groups on the surface of protein molecules. We have developed a maleimide surface based on the zero background PEG coating, as shown in Figure 1. The Maleimide derivatized surface covalently binds sulphydryl groups that become available in biomolecules after the reduction of disulphide bonds or after the chemical modification of primary amines through the introduction of SH groups with specific reagents. The PEG functionality ensures that binding of particular molecules to the surface is only through the specific interaction with the immobilized protein molecule during the assay and the commonly seen background problem is solved.

Fig. 1. The zero background maleimide surface consists of the reactive double bound, tethered to the high-density PEG coating. A protein molecule is attached via surface –SH group(s) but is otherwise repelled from the PEG coating. Excess -SH groups are easily de-activated in a simple washing step. The same immobilization reaction applies to peptides, antibodies, and oligonucleotides. Fig. 2. Fluorescence images of dye-conjugated oligo (-SH) and antibody microarrays fabricated (hand spotting) on maleimide-PEG/glass slides. The diameter of each spot is about 500 um.

To demonstrate the performance, we show in Figure 2 thiol-conjugated oligo microarrays (upper panel) and antibody microarrays (lower panel) fabricated on Mal/PEG/glass slides. Briefly, nanoliter droplets of dye-conjugated oligo (30 mers) or antibody solution containing 10% glycerol are deposited on the glass slide and incubated for 30 minutes. SH-groups in remaining area are removed by a deactivating buffer for 30 minutes at room temperature. The immobilized antibody on the surface is detected by 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. The Mal/PEG coated glass slides are ideal for biomolecules containing free –SH group (s). Disulphide bonds two cysteine residues in proteins are very stable, but can be reduced with reducing agents (R-SH), such as Dithiothreitol, 2-Mercaptoethanol, etc.. The non-fouling property of the high density PEG coating is critically important for maintaining protein activities at the surface and for minimizing non-specific adsorption of other abundant biomolecules in crude samples, like serum.

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 Mal/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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© 2014 MicroSurfaces, Inc. 2014