Typical methods for stable immobilization of proteins often involve time-consuming surface modification of silicon-based materials to enable specific binding, while the nonspecific adsorption method is faster but usually unstable. Herein, we fused a silica-binding protein, Si-tag, to target proteins so that the target proteins could attach directly to silica substrates in a single step, markedly streamlining the immobilization process. The adhesion force between the Si-tag and glass substrates was determined to be approximately 400-600 pN at the single-molecule level by atomic force microscopy, which is greater than the unfolding force of most proteins. The adhesion force of the Si-tag exhibits a slight increas... More
Typical methods for stable immobilization of proteins often involve time-consuming surface modification of silicon-based materials to enable specific binding, while the nonspecific adsorption method is faster but usually unstable. Herein, we fused a silica-binding protein, Si-tag, to target proteins so that the target proteins could attach directly to silica substrates in a single step, markedly streamlining the immobilization process. The adhesion force between the Si-tag and glass substrates was determined to be approximately 400-600 pN at the single-molecule level by atomic force microscopy, which is greater than the unfolding force of most proteins. The adhesion force of the Si-tag exhibits a slight increase when pulled from the C-terminus compared to that from the N-terminus. Furthermore, the Si-tag's adhesion force on a glass surface is marginally higher than that on a silicon nitride probe. The binding properties of the Si-tag are not obviously affected by environmental factors, including pH, salt concentration, and temperature. In addition, the macroscopic adhesion force between the Si-tag-coated hydrogel and glass substrates was ∼40 times higher than that of unmodified hydrogels. Therefore, the Si-tag, with its strong silica substrate binding ability, provides a useful tool as an excellent fusion tag for the rapid and mechanically robust immobilization of proteins on silica and for the surface coating of silica-binding materials.
