Biotinylation dendrimers

A similar type of biotin-dendritic multimer also was used to boost sensitivity in DNA microarray detection by 100-fold over that obtainable using traditional avidin-biotin reagent systems (Stears, 2000 Striebel et al., 2004). With this system, a polyvalent biotin dendrimer is able to bind many labeled avidin or streptavidin molecules, which may carry enzymes or fluorescent probes for assay detection. In addition, if the biotinylated dendrimer and the streptavidin detection agent is added at the same time, then at the site of a captured analyte, the biotin-dendrimer conjugates can form huge multi-dendrimer complexes wherein avidin or streptavidin detection reagents bridge between more than one dendrimer. Thus, the use of multivalent biotin-dendrimers can become universal enhancers of DNA hybridization assays or immunoassay procedures. 

Biotinylated dendrimers also have been used to develop targeting conjugates for therapeutic use in targeting cancer cells. Wilbur et al. (1998) studied several different PAMAM... 

Purify the biotinylated dendrimer by diluting it with an equal volume of water and then using dialysis, ultrafiltration, or size exclusion chromatography. 

The biotinylation of amine-dendrimers may be accomplished using either an organic reaction environment or an aqueous medium. For modification of PAMAM dendrimers with a biotinidase resistant biotin compound, Wilbur et al. (1998) performed the reaction in DMF with triethylamine as catalyst (proton acceptor). The following protocol illustrates this type of procedure using the biotinylation reagent NHS-PEG/pbiotin, which closely compares to the biotinidase insensitive compound used in the published procedure. 

Another method that can be used to biotinylate an amine-dendrimer is to do a similar reaction in aqueous buffer conditions. The following protocol is based on the methods of Tomalia et al. (1998) and Mamede et al. (2003). 

Add a 3-fold molar excess of biotinylation reagent over the molar quantity of dendrimer present. For the use of sulfo-NHS-LC-biotin (MW 556), this represents the addition of 2.1pmol or 1.16mg. This reaction ratio will result in a modification level of about 2.5 biotin groups per dendrimer. Other molar ratios also may be used, depending on the desired level of modification and the intended use for the conjugate. 

For instance, a dendrimer easily can be coupled with a large number of fluorescent dyes and still provide additional coupling sites for biotinylation. The only limitation to the number of fluorescent modifications is if fluorescence quenching starts to take place, in which case no further modifications will result in increased signal. A series of such conjugates using different levels of fluorophore modification should be done to determine the optimal level of dye-to-dendrimer before quenching occurs. 

Figure 7.21 Dendrimers that are fluorescently labeled as well as biotinylated create enhanced detection reagents for use in (strept)avidin-biotin-based assays. Large complexes containing multiple fluorescent dendrimers can bind to antigens and form a highly sensitive detection system that exceeds the detection capability of fluorescently labeled antibodies.

Wilbur, S.D., Pathare, P.M., Hamlin, D.K., Buhler, K.R., and Vessella, R.L. (1998) Biotin reagents for antibody pretargeting. 3. Synthesis, radioiodination, and evaluation of biotinylated starburst dendrim-ers. Bioconjugate Chem. 9, 813-825. 

Fig. 33.2. Schematic picture of the methods of immobilization of the aptamers (A) chemisorption. Biotinylated aptamer is immobilized on a surface covered by (B) avidin, (C) neutravidin and (D) avidin on a surface of poly(amidoamine) dendrimers (PAMAM). (E) Aptamer is immobilized on carbon nanotubes activated by Tween 20 (it is partially adapted from Ref. [39] with permission of American Chemical Society).

Scheme 3. Idealized sketch of a gold surface modified with a multilayered assembly prepared by (a) alternate deposition of layers of poly(acryhc acid) and poly-L-lysine on a positively charged cystamine SAM (the multilayer assembly is stabilized by electrostatic interactions between adjacent layers, [136]) and by (b) covalent attachment of a layer of G4 poly(amidoamine) dendrimer to a 11-mercapto-l-undecanoic acid SAM after activation of the surface-bound cEirboxylic groups with pentafiuorophenol and EDC. The dendrimer monolayer is afterwards biotinylated by covalent binding of desthiobiotin amidocaproate (not shown) to assemble an overlayer of avidin by affinity interactions [133].

Figure 13. (a) Formation of mixed SAM on Au electrode, (b) immobilization of Fc-D, (c) immobilization of thiolated capture probe with bifunctional linker (succinimidyl 4-(/V-maleimidomethyl)cyclohexane-l-carboxylate (SMCC)), (d) hybridization with target, (e) hybridization with biotinylated detection probe, (f) association with avidin-alkaline phosphatase, (g) description of the process of the electrocatalytic reaction of />-aminophenol (p-AP) via electronic mediation of ferrocenyl dendrimer (Adapted from Ref. [160])... 

Maximum Admissible Concentration (MAC) = 0.1 og/L) for pesticides in drinking water. Recent studies have been aimed at increasing the sensitivity of CMIA by attaching several M-CO probes borne by PAMAM dendrimers to antibodies [19] or to avidin which can then be used in conjugation with biotinylated antibodies [18]. Detection of these labeled antibodies can be carried out on membranes, making it possible to omit the step of extracting the free fraction by solvent. 

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