Streptavidin binding

Fig. 57. DNA-nanoparticle assembly based on biotin-streptavidin binding. Discrete, A and B, and extended, C, networks have been constructed. Adapted from Ref. 186).

On the other hand, the volume effects provided by e.g. hydrogels such as dextran layers14, 15 will allow an increased number of binding sites. In addition, Figure 9 shows that biotin streptavidin binding and the use of HIS-... 

Fig. 9.19 Resonace wavelength shift during streptavidin binding to a ring resonator sen sor surface. The concentration of strepatavidin is increased in one decade steps during the experiment...

This method, either standard or elite (increased molar ratio), remains the forefront of much of the immunocytochemistry being performed today, and will be the main subject of this chapter. There are new methods, though, that are being used with increased frequency, such as the labeled-avidin binding method, sometimes called the streptavidin-binding method, and a newer catalyzed amplification method that uses avidin, biotin, peroxidase, and a biotinyl tyramide to achieve even more sensitivity. These methods will be discussed at the end of this chapter. 

The use of biotinylated antibodies provides perhaps the greatest versatility and sensitivity of all methods The affinity of biotin for avidin or the more usually used streptavidm is very high, and the latter can be conjugated to radioisotope, fluorescent moiety, or enzyme Again, the basic procedures are the same as outlined for l25I-labeled antibodies with the additional steps required for streptavidin binding and subsequent incubation with enzyme substrate (see also Chapters 17 and 18)... 

A wide range of immobilization chemistries are commercially available in conjunction with Sepharose beads. We have investigated a limited subset of these possibilities which include direct, nonoriented immobilization via Schiff s base chemistry, oriented nonco-valent immobilization via immobilized metal affinity chromatography resins and oriented noncovalent immobilization via biotin-streptavidin binding. At present we favor direct, covalent attachment of proteins via primary amines since it is highly efficient (typically better than 85% yield), minimizes leaching and provides the best NMR results (Figure 6.2). 

FIGURE 6.35 Schematic of the experimental protocol for streptavidin affinity chromatography. (1) The channel is initially filled at room temperature with a suspension of biotinylated, PNIPAAm-coated beads (100 nm). (2) The temperature in the channel is then raised to 37°C, and the beads aggregate and adhere to the channel walls. (3) Buffer is then pumped through the channel (the presence of flow is indicated in this schematic by an arrow), washing out any unbound beads. (4) A fluorescently labeled streptavidin sample (2.5 pM) is then introduced into the flow stream. (5) Streptavidin binds to the beads, and any unbound streptavidin is washed out of the channel. (6) Finally, the temperature is reduced to room temperature, leading to the breakup of the bead aggregates. Beads, bound to labeled streptavidin, elute from the channel [203], Reprinted with permission from the American Chemical Society. 

Fig. 13 SPR and AFM analyses of controlled streptavidin recognition on mixed oligo(ethylene glycol) layers, (a) Chemical structure of the mixed layer components linear oligo(ethylene glycol) left) and biotinylated moiety (right), (b) Streptavidin binding at 23°C and (c) at 45°C. (d) SPR sensogram of streptavidin adsorption at 23 and 45°C. Arrows show injections of (a) water (b) streptavidin solution and (c) subsequent water rinse at 23°C and (d) streptavidin solution and subsequent water rinse at 45°C. (e) Tapping mode AFM images in liquid phase after injection of streptavidin solution at 23°C and (f) at 45°C. Reprinted, with permission, from [185]. Copyright (2008) American Chemical Society...

The strong and specific biotin-streptavidin binding was used to assemble biomolecule-functionalized nanoparticles in multilayered structures.67 Application of an electrical field allowed the assembly of multilayer structures by using extremely low concentrations of nanoparticles with minimal nonspecific binding. A microelectrode array was used to facilitate the rapid parallel electrophoretic transport and binding of biotin- and streptavidin-functionalized fluorescent nanoparticles to specific sites. By controlling the current, voltage, and activation time at each nanoparticle adsorption step, the directed assembly of more than 50 layers of nanoparticles was accomplished within an hour. 

The concept of MW enhancers was developed to increase polarization value changes caused by large size differences between substrate and product when small peptide substrates were used. The most frequently used approach is based on the attachment of a biotin molecule to the terminus of the peptide on the opposite site of the scissile bond to the fluorophore. After the incubation of the substrate with the protease, streptavidin is added to the assay. Streptavidin binds to biotin and enhances the masses of the substrate and the unlabeled product, thus causing an increase of the polarization... 

Fig. 3 Biotin and biotin derivatives, a Valeric arm allows addition of various groups without interfering with streptavidin binding...

Pirrung and Huang [107] synthesized a biotin derivative with an attached 350 nm-sensitive nitrobenzyl group that prevented streptavidin binding, and a 6-aminocaproic acid linker terminated in an active NHS ester (MeNPOC-... 

A DNA optical sensor system was proposed by Cass and co-workers [35] based on the combination of sandwich solution hybridization, magnetic bead capture, flow injection and chemiluminescence for the rapid detection of DNA hybridization. Sandwich solution hybridization uses two sets of DNA probes, one labelled with biotin, the other with an enzyme marker and hybridization is performed in solution where the mobility is greater and the hybridization process is faster, rather than on a surface. The hybrids were bound to the streptavidin-coated magnetic beads through biotin-streptavidin binding reaction. A chemiluminescence fibre-optic biosensor for the detection of hybridization of horseradish peroxidase-labelled complementary DNA to covalent immobilized DNA probes was developed by Zhou and co-workers [36]. 

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