Thrombin signal aptamer

Figure 36 The structure-switching signaling aptamer. A DNA duplex composed of three strands of DNA places a fluorophore (F) close to a quencher group (Q). Upon addition of thrombin, the QDNA piece is released, and the fluorescence increases (See ref 67).

A similar approach was conducted by using quantum dots (QDs) for signal amplification [51]. In this case the hybrid formed by a thiol-labeled oligonucleotide and the thrombin aptamer was immobilized onto a gold electrode. When binding to thrombin the aptamer adopts its G-quartet structure and only the single-stranded probe remained onto the electrode, which is now available for hybridization with a QD-labeled complementary oligonucleotide (Fig. 2.11). 

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. 

The limitation of this signal-off architecture was improved in another work [49] in which the aptamer was first allowed to interact with short partially complementary nucleotide modified by MB. (MB)-tagged oligonucleotide with aptamer formed rigid duplex that prevents the MB tag from approaching the electrode surface, so the reduction current is suppressed. The addition of thrombin resulted in quadruplex formation and approach of MB to the surface of electrode. Thus, the amplitude of reduction signal increased (Fig. 33.3D). This method allowed to detect thrombin at concentrations as low as 3 nmol/L. 

Fig. 3 Electrochemical aptamer-based sensor of redox-tagged DNA against specific targets, (a) When the aptamer comes in contact with a small molecule, in this case cocaine, it folds, and the redox tag is brought closer to the electrode, increasing the current, (b) When the aptamer comes in contact with thrombin, the tag moves away from the surface, decreasing the electrochemical signal. Reproduced from [85] with permission. Copyright Langmuir, 2007...

To study the reproducibility and the reusability of the sensor, successive washing steps were performed with solutions of 8 M Urea and 0.2 M Ca EDTA followed by re-incubation with 10 nM thrombin. Urea was chosen to destabilize the protein aptamer complex and Ca /EDTA was chosen to disrupt the G-quadruplex. Spectra recorded after washing exhibit the low intensity unbound signal and signal increases repeatedly after incubation with 10 nM thrombin - these two states can be... 

The analytical data, expressed as frequency shift, are the differences in the frequency of the crystal before the addition of thrombin and after the washing with buffer subsequent to the affinity interaction. A signal generated by the aptamer-protein interaction is considered significative when the difference between the frequency values corresponding to the two buffers is higher than 3 Hz. Different concentrations of the protein can be used to build a calibration plot. An example of calibration plot is shown in Fig. 5. 

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