The covalent coupling of alkynylated proteins to functionalized carriers using the Click Azide Alkyne Cycloaddition

Abstract

Towards the development of innovative biosensors and bio-functionalized carriers, improved protein immobilization methods based on stable covalent bonds between the protein and carrier surface to replace the frequently used physical adsorption are becoming more important. Click chemistry, e.g. the copper catalyzed alkyne azide cycloaddition (CuAAC), offers opportunities to realize this goal. Ellipsometry is used in this study for the assessment of the amount of protein coupled to the surface. This technique allows the detection of layer thicknesses with a subnanometer precision and measures surface mass with an accuracy of 1 ng/cm2. In this study, two model proteins, Protein A and Maltose Binding Protein (MBP), were coupled covalently to a carboxylated silicon carrier. Protein A was coupled by a non-oriented (random) coupling procedure via the natural amino groups of the lysines in the amino acid sequence. Hereto, a (bio-orthogonal) alkyne functionalized NHS ester was used to derivatize 10 out of 63 lysines present in protein A by EDC/NHS coupling. On the surface side, a 3-azido-1-aminopropane linker was attached to the carboxylated silicon carrier using EDC/NHS. This allows covalent protein-carrier coupling by CuAAC. However, large improvement in reproducibility, sensitivity and specificity can be expected when the proteins are not only covalently coupled to the carrier but also are homogeneously oriented on the surface. This can be achieved by introducing a bio-orthogonal functional group at a single, site-specific location in the protein. Here we present MBP which was, via an in vitro modification technique, site-specifically functionalized with an alkyne group. By means of the procedure described above for Protein A, it was covalently and oriented coupled to the complementary azide-functionalized carrier. By using antibodies against these model proteins, the activity/specificity of the proteins after immobilization has been verified and quantified using ellipsometry. As compared to physical adsorption based methods, the assessed protein/antibody quantities were significantly higher, confirming the proof of principle. The combination of site-specifically functionalized proteins, Click chemistry and ellipsometry paves the way for further innovative improvements in the field of biosensors and bio-functionalized carriers.