Thrombin electrochemical aptasensor

Figure 12.18 (a) Electrochemical aptasensor for thrombin based on the control of electron... 

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.

Sandwich-type sensing platforms are also nsed widely in electrochemical aptasensors (Willner and Zayats, 2007), especially for common model molecnles such as a-thrombin (Mir et al., 2006 Polsky et al., 2006 Centi et al., 2007) and platelet-derived growth factor (PDGF) (Zhou et al., 2007), which possess two active aptamer-binding sites. This type of sensor usually... 

Ikebukuro et al. [12] first reported an electrochemical aptasensor for the detection of thrombin based on a sandwich-based assay. Two different aptamers specific for thrombin were used the 29-mer thiolated aptamer and the 15-mer aptamer labeled with glucose dehydrogenase [GDH]. The thiolated aptamer was immobilized onto gold electrodes thrombin at different concentrations and then the enzyme-labeled aptamer was added to the aptamer-modified electrodes. The electric current generated by the addition of glucose was measured at 0.1 V vs. Ag/AgCl in a buffer containing... 

Centi et al [18] described various approaches for the development of electrochemical aptasensors for the detection of thrombin using magnetic beads as solid support and carbon... 

Another example of electrochemical aptasensor based on Au-NPs as labels for the detection of thrombin is reported by Zheng et al. [28]. The assay was based on a sandwich format, in which the aptamerl (15-mer DNA aptamer with an amino group at its 5 end) was immobilized onto carboxyl functionalized magnetic beads. Such aptamer-coated magnetic beads were used for capturing and separation. Thrombin and Au-NP-labeled aptamerll were then added to... 

A detection limit of 0.55 fM of thrombin was calculated. Authors attribute the significant improvement of the sensitivity of such aptasensors with respect to others present in literature to the use of a nanoelectrode, formed by immobilization of Au-NPs on the surface of a gold electrode, to the use of NPs as labels, and to the use of DPV technique for the detection of the dissolved Cd + in the solution. Moreover, the electrochemical aptasensor was successfully tested in some serum samples. 

DNA probes, the first one linked with paramagnetic beads and the second one modified with Au-NPs via biotin-streptavidin affinity reactions. Zheng et al. [67] employed this detection methodology for the development of a specific electrochemical aptasensor for the detection of thrombin. The sensor was based on a sandwich format of magnetic nanoparticle/thrombin/Au-NP and signal amplification by forming network-like thiocyanuric acid/Au-NPs. A detection limit of 7.82 aM was achieved. 

As an example of application of electrochemical aptasensor to medical diagnosis. Sun et al. developed a label-free electrochemical aptasensor for thrombin detection in whole blood. For the fabrication of this aptamers, self-assembled multilayers of carboxymethyl-polyethylene glycol-carboxymethyl (CM-PEG-CM) and thrombinbinding aptamer (TBA) were used. The thrombin molecules in the medium bound onto the TBA which leads to current decrease. This current decrease was monitored by differential pulse voltammetry. A linear range of 1 pM to 160nM was obtained for thrombin with LOD value of 1.56 x (Sun et al., 2013). 

Sun, C., Wang, X., Yang, X., Xing, L., Zhao, B., Yang, X., Mao, C., 2013. A label-free electrochemical aptasensor for sensitive thrombin detection in whole blood. Electrochimica Acta 106, 327-332. 

Zhao, J., Zhang, Y, Li, H., Wen, Y, Fan, X., 2011a. Ultrasensitive electrochemical aptasensor for thrombin based on the amplification of aptamer-AuNPs-HRP conjugates. Biosens. Bioelectron. 26, 2297—2303. 

Electrochemical methods of detection affinity interactions at the surfaces are rather effective due to their relative simplicity and low cost. Amperometric aptasensor based on sandwich assay was proposed by Ikebukuro et al. [42]. They used two aptamers selective to thrombin. 

Mir, M., Vreeke, M., Katakis, I. (2006). Different strategies to develop an electrochemical thrombin aptasensor. Electrochem. Commun. 8, 505-511... 

Figure 3.4 Amplified electrochemical analysis of thrombin using (A) thrombin-catalyzed generation of the electroactive substrate (3) (B) and (C) enzyme-tethered anti-thrombin aptasensors as amplifying labels (HRP and GDH, respectively).

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). 

An amplified electrochemical impedimetric aptasensor for thrombin has been also described [70]. A nice improvement in the detection sensitivity was achieved by constructing a sandwich platform where the thiolated aptamers were immobilized on a gold substrate to capture the thrombin molecules. Then, aptamer functionalized Au-NPs were used to amplify the impedimetric signals (Fig. 4.11). A detection limit of 0.02 nM, with a linear range of 0.05 to 18 nM was achieved. 

Bai, H.-Y., del Campo, F.J. and Tsai, Y.-C. (2013) Sensitive electrochemical thrombin aptasensor based on gold disk microelectrode arrays. Biosens. Bioelectron., 42,17. 

Aptamer-based biosensors, also called aptasensor have gain a wide interest in the last years due to the advantages of aptamers compared to antibodies. Similar to antibodies, a variety of immobilization methods is available to bind aptamers to the sensor element. Aptasensors can be coupled to an electrochemical, optical or mass-sensitive transducer [13]. One of the successful examples for aptasensor was the detection of thrombin which was widely investigated [14]. Xiao et al. [15] have made an interesting development a redox compound (methylene blue) was inserted into the thrombin aptamer. When the target bound to the aptamer, the induced conformation change inhibited the electron transfer from the methylene blue to the electrode. This change could be detected amperometrically. 

---