Biotinylated aptamer

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).

The biotinylated aptamer (0.5 pM, (see Note 3)) is thermally treated before its immobilization. The thermal treatment can unfold the aptamer strand making the biotin label at the 5 end available for the interaction with streptavidin on the chip surface. Before the immobilization, the biotinylated aptamer is heated at 90°C for 1 min to unfold the DNA strand and then cooled in ice for 10 min to block the DNA in its unfolded structure (44) (see Note 4). 

The biotinylated aptamer was easily immobilized on a streptavidin polystyrene-divnyl benzene support. Enantiomers of small biomolecules were also resolved efficiently (Michaud et al., 2004). To develop this strategy on a large scale, several problems have to be solved, notably the cost and the problem of the aptamer stability if samples contaminated with nucleases are used. Solutions discussed above (modified oligonucleotides) can be considered. 

Efficient immobilization of aptamers on surfaces is necessary for the construction of tongh, stable sensors and assay systems as one necessary step to overcome limitations for practical applications (Bini et al., 2007). Bini et al. (2007) have compared thrombin aptamers immobilized on a gold snrface by chemisorption (thiolated aptamer) and by biotin-streptavidin interaction (biotinylated aptamer carrying a linker) on a gold surface modified by a thiol-dextran-streptavidin layer. The linker-modified aptamer immobilized via streptavidin-biotin showed better reproducibility and sensitivity results for the quartz crystal sensor. Aptamers can be used for the functionalization of titanium-alloy surfaces (e.g., implant material, scaffolds) to enhance cell adhesion. The aptamers directed to osteoblasts are fixed electrochemically on the snrface of the alloy and promote cell adhesion (Gno et al., 2005, 2007). 

Figure 8.3 Sandwich assay developed for thrombin using two different aptamers, magnetic particles, and enzymatic amplification. Two selected aptamers binding thrombin in two different, nonoverlapping sites are used. The protein captured by the first aptamer fixed onto magnetic particles is detected after addition of the second biotinylated aptamer and of streptavidin labeled with an enzyme (alkaline phosphatase). Detection of the product generated by the enzymatic reaction is achieved by differential pulse voltammetry onto screen-printed electrodes onto which the magnetic nanoparticles are deposited and kept in contact through a magnet. [From (Centi et al., 2007b).]...

Q Streptavidin-coated magnetic bead I 5 biotinylated aptamer... 

A disposable electrochemical competitive assay for detection of IgE was proposed by Papamichael et al. [16]. In this work the IgE antigen was immobilized on the surface of screen-printed electrodes, then a competition step between IgE bound to the electrode surface and IgE in solution for the biotinylated aptamer was left to occur. At this point the streptavidin-alkaline phosphatase conjugate... 

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