Protein Layers Streptavidin

Standard CM-AFM using a liquid cell filled with Tris buffer solution is carried out with oxide sharpened tips a cantilever spring constant of 0.1 N/m is used. The engagement procedure is carried out as described in Sect. 3.2. Similar to all 

Depending on the concentration of DNA used in the sample preparation procedure, the surface coverage can be adjusted. The fixation using the silanization procedure is beneficial in effectively immobilizing the DAN for AFM work. Stability in excess of 5 h has been reported [106] (Fig. 3.50). 

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Guided mode calculations were also carried out to compare the sensor response of several waveguide systems. In these simulations a model molecular monolayer is represented by a 2-nm thick layer with a refractive index of n 1.5. The optical properties of this model layer are typical of a dense layer of organic molecules on a substrate1 41, and are a reasonable approximation for a streptavidin protein layer bound to a biotinylated surface, the experimental model system we use to characterize our sensors. The ambient upper cladding was assumed to be water with a refractive index of n 1.32. For all examples, the lower cladding was assumed to be Si02 with an index of n 1.44. In the simulations, the effective index of... 

Surface immobilization of the capture molecules follows standard procedures that are commonly practiced in many biosensor applications and some are discussed in the previous section. Layers of carboxymethyl dextran. Protein A or Protein G, streptavidin-coated surface, or EDC [N-ethyl-N-(diethylaminopropyl) carbidimide]/NHS (N-hydroxysuccmimide)-based amine coupling through amide bond are used for protein (antibody, receptor, etc.) cross-linking. 

Ringsdorf and coworkers used monolayer techniques for the creation of lipid functionalized streptavidin layers.252,253 In their pioneering work, a monolayer of biotinylated phospholipids initially created a two-dimensional docking matrix for the immobilization of streptavidin. In an effort to further expand this system while maintaining control over its structure, bis-biotinylated bifunctional linkers were employed. Addition of these linkers to concanavalin A created a new protein-based bidentate linker. Upon addition to the monolayer of the lipid-functionalized streptavidin a second biotilynated protein layer was created. Upon the further addition of streptavidin, they achieved in a step-by-step construction process well-defined alternating protein triple layers of streptavidin and concanavalin A (Figure 7.37b). A similar approach has more recently been used to crosslink... 

Figure 6.5 Biotin head groups of a surface monolayer bind strongly to the protein streptavidin containing four biotin binding centres. A crystalline protein layer can now be observed by electron microscopy. The other binding centres can now be used to form a third monolayer with biotinylated proteins, e.g. the FAB fragment of F immunoglobulins. ...

These findings led us to a strategy for optimized protein binding even at well-ordered monolayers by using binary mixtures of biotin -and OH -terminated self-assembly systems. Figure 3 shows the surface-plasmon optical results of the thickness determinations for co-adsorbed SAM s and of their streptavidin binding. The striking result is the maximum protein layer thickness at ca 5-10 mole% biotinylated thiol indi-... 

Another interesting application of specific interactions in SAMs is the use of ligand-derivatized thiols to build up complex biosupramolecular architectures via molecular recognition-driven processes. Typical examples include the use of biotin- and mannose-terminated SAMs to biorecognize and assemble streptavidin and concanavalin A protein layers, respectively, on metal surfaces with diverse purposes (Figure 12). " ... 

Fig. 14 A schematic illustration of an orientated immobilization strategy via crystallized S-layer proteins a side view of the crystalline ordered S-layer proteins fused with the recognition center streptavidin for biotinylated biomolecules probes b front view of the orientated and crystalline ordered S-layer proteins...

Fig. 4 Macromolecular bioconjugates created using bifunctional streptavidins. Here, a biotinylated single-stranded DNA is bound to immobilized streptavidin. A complementary strand covalently attached to streptavidin is hybridized to the immobilized DNA, and a biotinylated protein is bound to the floating streptavidin layer...

Figure 21, Images (a,c) and Fourier transforms (b,d) of helical crystals of streptavidin formed on lipid tubules containing DODA-EOa-biotin (50). (a,c) Stain striations extend along the tubules. Protein densities are particularly visible at tube edges, corresponding to streptavidin molecules viewed edge-on. Scale bar 40 nm. (b,d) Distribution of Fourier transform amplitudes from the tubes shown in (a,c) corresponding to about 1700 streptavidin molecules. The fine spacing between layer lines indicates a helical repeat of 47 nm. Visible diffraction peaks extend up to 1.7 nm (arrowhead in (b)]. Reproduced from ref. 242 (Ringler et al., Chem. Eur. J. 1997, 3, 620) with permission ofWiley-VCH.

In addition to the solution-phase protein detection assays such as biobarcode assays, a surface-based protein detection method was developed by Niemeyer and Ceyhan (70). In this work, a biotin-labeled antibody was conjugated to DNA using streptavidin as a tinker molecule. This conjugate was hybridized to complementary DNA-AuNPs and antibody-functionalized AuNPs. These particles are allowed to form a sandwich structure with a target protein and a second antibody immobilized on a flat surface. This was followed by a silver enhancement step catalyzed by the AuNPs which resulted in a LOD of 50 fmol (200 pM). This method is conceptually similar to the corresponding surface-based scanometric detection of DNA. Further work demonstrated a similar protein detection scheme without the silver enhancement (71). Instead, multiple layers of secondary DNA-AuNPs were used to increase signal enhancement [LOD = 0.1 finol (2 pM)]. 

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