Thrombin electrochemical detection

Figure 12.19 Electrochemical detection of thrombin by the interaction between a nucleic acid and a redox-active oligothiophene polyelectrolyte. The electrical contact between the polyelectrolyte and the electrode by means of the aptamer/thrombin complex is blocked. Parts Reprinted with permission from Ref. 66b. Copyright Wiley-VCH Verlag GmbH Co.

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. 

In the past few years a new direction in TBA-based biosensors has been the development of methods for the electrochemical detection of thrombin. Some of the reported advantages of these electrochemical biosensors are their potential to provide high sensitivity, fast response times, low costs, easy fabrication, and the possibility for miniaturization. Ikebukoro and colleagues were the first to report on a TBA-based electrochemical sensor. Like others. 

Figure 39 Electrochemical detection of thrombin through the coupled oxidation of glucose by glucose dehydrogenase (See ref 71).

Figure 3.6 Electrochemical detection of thrombin by interaction between nncleic acid and a redox-active oligothiophene polyelectrolyte (4). (A) Blocking the electrical contact between the polyelectrolyte and the electrode by means of a aptamer-thrombin complex. (B) Separation of the aptamer-thrombin complex by the formation of a PNA-opened aptamer dnplex on the electrode and its analysis by the redox-active polyelectrolyte. (C) Voltammetric response to the analysis of different concentrations of thrombin according to method B (a) OM (b) 125nM (c) 250nM (d) 500nM, 1 p,M (f) 2 p,M. (Reprinted with permission from Floch et al., 2006. Copyright 2006 American Chemical Society.)...

An aptamer-based sandwich assay with electrochemical detection for thrombin analysis in complex matrixes using a target-capturing step by aptamer-functionalized magnetic beads was recently reported (Centi et al., 2007). The aptamer-sensing layers were fabricated on a surface of screen-printed electrodes. The high sensitivity of this sensor was demonstrated in the analysis of thrombin in buffer, spiked serum, and plasma. The concentrations detected by the electrochemical assay were in agreement with simulation software that mimics the kinetics of thrombin formation. 

An interesting approach based on the use of aptamer-functionalized platinum nanoparticles has recently been optimized (Polsky et al., 2006). In this case the metallic nanoparticles were used not as mere labels for electrochemical detection, but as catalytic molecules. This with the aim of overcoming some of the drawbacks associated with the thermal and environmental instability of traditional biological redox catalytic labels, such as enzymes. By following this approach the amplified electrochemical detection of thrombin was achieved by functionalizing platinum nanoparticles with the specific aptamer (Figure 8.2). 

Some papers have used the catal3 ic activity of thrombin for the determination of this protein. a-Human thrombin is a highly specific serine protease that catalyses the hydrol is of the thrombin chromogenic substrate, j8-Ala-Gly-Arg-p-nitroaniline producing p-nitroaniline. The rate of yellow colored p-nitroaniline formation can be followed by its UV absorption at 405 nm, or electrochemi-cally by the reduction of its nitro group. The electrochemical detection offers benefits in terms of sensitivity and speed. When saturated by enzyme substrate the formation rate of p-nitroaniline is proportional to the enz3mie concentration. 

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. 

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... 

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. 

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