Click chemistry on surfaces

Application note: Click Chemistry on the Zero Background Alkyne/PEG Surface

Since its introduction by Sharpless almost 10 years ago [Angew. Chem. Intern. Ed. 2001, 40, 2004], the so-called “Click Chemistry” has been developed into a versatile tool for the conjugation of a wide variety of organic, polymeric, and biological molecules. This process requires a highly selective and facile conjugation reaction between two small chemical groups under benign reaction conditions, such as the most popular Huisgen cycloaddition reaction between azide and alkyne in aqueous solution catalyzed by Cu(I). This powerful method has also been demonstrated for the conjugation of organic and biomolecules to solid surfaces. In response to popular demand, we have recently extended our ZeroBkg® line of surfaces to include the powerful Click chemistry, Fig. 1. We start with our high density PEG coating and covalently attach alkyne groups at controlled density to the PEG surface. Users of this surface coating product can then easily attach azide-conjugated molecules, such as proteins or glycans, to the PEG brush surface. The hydrophilic PEG brush ensures exceptionally low background and high activity of the immobilized probe molecule.

To demonstrate the performance, we show in Fig. 2 the specific immobilization of an azide-conjugated protein, fibrinogen. As negative control, we show the absence of adsorption for same protein without the azide-tag. Here detection of adsorbed protein was achieved by primary antibody, followed by CY3-labeled secondary antibody. We draw two important conclusions from these results: 1) the conjugation of the azide-tagged protein to the alkyne presenting surface is highly efficient; 2) the PEG coating remains repulsive to protein molecules without the azide tag, thus ensuring high selectivity and low background. No blocking is involved! Combining the Click Chemistry with the low background PEG brush allows us to offer a powerful product. Users of these surfaces can easily immobilize a variety of biomolecules, such as proteins, glycans, and DNA.

click chemistry on surface

Figure 1. The zero background alkyne surface consists of the reactive terminal alkyne groups tethered to the high-density PEG coating. A protein molecule is attached via azide (-N3) group(s) but is otherwise repelled from the PEG coating. The same immobilization reaction applies to peptides, antibodies, glycans, small molecules, and oligonucleotides.

alkyne azide

Figure 2. Fluorescence images of azide-conjugated fibrinogen (four spots on the left) covalently attached to the alkyne/PEG surface via Click Chemistry. The left part shows four spots from negative control, i.e., the adsorption of fibrinogen without azide-label. The spots of immobilized fibrinogen were obtained from hand spotting (pippeting) on the alkyne/PEG/glass slides. The diameter of each spot was about 1 mm. Detection was achieved by primary antibody followed by CY3-labeled secondary antibody.

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 alkyne/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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