Protein-aptamer interactions electrochemical methods

DETECTION OF PROTEIN-APTAMER INTERACTIONS BY MEANS OF ELECTROCHEMICAL INDICATORS AND TRANSVERSE SHEAR MODE METHOD... 

In this chapter the focus is on the use of electrochemical indicators and of the TSM method for the study of protein-aptamer interactions. Aptamers sensitive to thrombin are used for the fabrication of biosensors. Attention is given to methods of immobilization of aptamers to a solid support and how this affects the interaction of thrombin with the aptamer. The effects of aptamer structure, of the presence of the thrombin inhibitor heparin, and of the ions and pH on the binding properties of aptamer are presented. The advantages of electrochemical indicators and the TSM method for the detection of thrombin-aptamer interactions are demonstrated. 

In its simplest, QCM, format, protein-aptamer interactions were analyzed by Liss et al. (2002). They compared the interaction of IgE with DNA aptamer as well as with anti-IgE antibodies. Although the detection limit was similar in the two cases, the advantage of the aptasensor was its possibility of surface regeneration, which was impossible for an antigen-based biosensor. However, recently it has been shown that immobilization of anti-IgE on the dendrimer surface also allows us to regenerate an immunosensor (Svobodova et al., 2006). The QCM method was recently compared with the electrochemical biosensor assay of thrombin detection (Hianik et al., 2005, 2007). It has been shown that the sensitivity of thrombin detection was similar for the two methods. Mascini and co-workers showed that similar results in sensitivity and selectivity in the detection of Tat peptide with RNA aptamer can be obtained by the QCM and SPR methods (Tombelli et al., 2005b). 

Electrochemical indicator methods are based on the application of redox probe that undergoes oxidation and reduction transition due to electron transfer from electrode surface to a probe. In 2005, several studies that used methylene blue (MB) as an electrochemical indicator were published. MB is positively charged low-molecular-weight compound that can be reduced by two electrons to a leucomethylene blue (LB). The reduction process can be effectively monitored, e.g., by differential pulse voltammetry or coulometry. In presence of redox probe Fe(CN)6, the LB is oxidized to MB and the system is regenerated [44,45]. In papers by Hianik et al. [31,46], MB was used as the indicator of detection of interaction of human thrombin with DNA aptamer. The method of detection is schematically shown in Fig. 33.3B. MB binds both to DNA and to the protein. For charge transfer from electrode to MB, i.e., for MB reduction, it is important that MB should be close to the electrode surface. Therefore, the charge transfer from the electrode... 

Hianik, T., Ostatnd, V., Zajacovd, Z., Stoikova, E., Evtngyn, G. (2005). Detection of aptamer-protein interactions using QCM and electrochemical indicator methods. Bioorg Med Chem Lett 15, 291-295. 

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