Thrombin aptamer

Several electrical aptamer biosensors implemented the biocatalytic hydrolytic activities of thrombin, or the fact that proteins (e.g., thrombin) often include several binding sites for the formation of supramolecular complexes with different aptamers. The bioelectrocatalytic detection of thrombin by an electrical aptasensor was demonstrated by formation of an aptamer-thrombin complex on the electrode, followed by a thrombin-mediated hydrolysis of the nitroaniline-functionalized peptide, (22), yielding the redox-active product nitroaniline, (23), which was analyzed electrochemically76 (Fig. 12.20b). A further bioelectrocatalytic aptasensors configuration is depicted in Fig. 12.20c, where the multidentate formation of aptamer-protein supramolecular complexes was used to analyze thrombin.76 Thrombin includes two different binding sites for aptamers.77 One of the thrombin aptamers... 

Fig. 8.2 Nyquist plot of impedimetric sensor (Section 8.4.3.3). The redox couple ferro/ ferricyanide recorded at (a) bare Au electrode (b) Au electrode modified with thrombin-binding aptamer (c) Au/aptamer electrode with added 2-mercapto ethanol and (d) electrode with bound complementary thrombin/aptamer complex. Each plot was obtained separately (adapted from Radi etal.,2005)...

Two different formats of electrochemical detection of thrombin-aptamer interactions using MB were recently developed [32,49]. The aptamer was modified at one end by thiol group and at the other end by MB. Without thrombin, the MB possesses the reduction signal. Addition of thrombin shifted equilibrium from unfolded to folded aptamer conformation. This resulted in an increase in distance of MB from the electrode. As a result the reduction signal decreased. 

Latham, J.A., Johnson, R. and Toole, J.J. (1994) The application of a modified nucleotide in aptamer selection novel thrombin aptamers containing 5-(l-pentynyl)-2l-deoyuridine. Nucl. Acids Res., 22, 2817-2822. 

Sumikura, K., Yano, K., Ikebukuro, K. and Karube, I. (1997) Thrombin-binding properties of thrombin aptamer derivatives. Nucleic Acids Symp. Ser., 257-258. 

Figure 8.14 Scheme depicting the analytical procedure for biodetection based on catalytic H202 reduction at PtNPs (a) Detection probe modified PtNPs for DNA detection (b) thrombin aptamer DNA sequence modified PtNPs for thrombin protein detection.69 (Reprinted with permission from R. Polsky et al., Anal. Chem. 2006, 78, 2268-2271. Copyright 2006 American Chemical Society.)... 

The suitability of aptamers as in vivo imaging agents is demonstrated in references [39-41], who used anti-neutrophil elastase and anti-thrombin aptamers for imaging of inflammation and thrombus in vivo, respectively. 

DNA- binding aptamers have been designed to bind to mRNA, g-quad-ruplexes, Tenascin-C, a protein found in the tumor matrix, and to thrombin. Aptamers have been selected as inhibitors of HIV-1 integrase, human RNase Hl, human pro-urokinase " and for the design of molecular beacons. An allosteric aptamer has been designed for binding as a colorimetric probe for cocaine. ... 

Figu re 3.4 Amplified detection of thrombin on surfaces by the catalytic enlargement of thrombin aptamer-functionalized GNPs. (Reprinted with permission from Reference [17], Copyright 2007 American Chemical Society.)... 

This chapter deals with the coupling of the thrombin aptamer with quartz crystal microbalance devices for the development of aptamer-based piezoelectric biosensors. 

Holland, CA., Henry, A.T., Whinna, H.C., Church, F.C. (2000). Effect of oligodeoxy-nucleotide thrombin aptamer on thrombin inhibition by heparin cofactor II and antithrombin. FEBS Lett. 484, 87-91... 

Heckel, A., Mayer, G. (2005). Light regulation of aptamer activity an anti-thrombin aptamer with caged thymidine nucleobases. J Am Chem Soc 127, 822-823. 

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 3.3 Electrochemical aptasensor for thrombin based on the control of electron transfer between redox-labeled aptamer and the electrode. (A) Controlling the orientation of the redox label with respect to the electrode upon formation of a thrombin-aptamer complex. (B) Differential pulse voltammetry corresponding to an analysis of different concentrations of thrombin by a ferrocene-tethered aptamer (a) 0, (b) 10, (c) 20, and (d) 30 nM. (Reprinted with permission from Radi et al., 2006. Copyright 2006 American Chemical Society.) (C) Activation of the electrical contact of methylene blue-tethered aptamer upon formation of the respective aptamer-thrombin complex. (D) Voltammo-grams corresponding to analysis of the thrombin by the configuration depicted in part (C) curves (a) no thrombin (b) thrombin 10 nM (c) thrombin 256 nM. (Reprinted with permission from Xiao et al., 2005. Copyright 2005 American Chemical Society.) (E) Blocking the electrical response of methylene blue intercalated into the stem of a DNA hairpin as a result of formation of an aptamer-thrombin complex.

Similarly, an electrode modified with a thrombin aptamer containing a redox label on its free end creates a steady current in the absence of the target (Xiao et al., 2005). When thrombin is added, the aptamer structure forms and removes the label from the surface, thus interrupting the electron transfer (Figure 4.6). 

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. 

Another method of detection of the thrombin-aptamer interaction was proposed by Bang et al. (2005). They immobilized aptamer beacon modified by an amino group to a self-assembled monolayer on a gold surface formed by... 

Recently, we applied the TSM sensor for the characterization of all steps in the sensor fabrication and detection of thrombin-aptamer interactions (Grman et al.,... 

Kankia, B. T, Marky, L. A. (2001). Folding of the thrombin aptamer into a G-quadruplex with Sr + stability, heat, and hydratation. J Am Chem Soc 123, 10799-10804. 

Hamaguchi et al. (2001) have constructed a thrombin detection system using a designed thrombin aptamer and fluorescence resonance energy transmission (FRET). The oligonucleotides complementary to the nucleotides at the 3 end... 

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