Signaling aptamers interaction detection

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. 

FRET to occur, and thus no FI emission is observed. The specificity of this assay was also examined for mixed samples. The mixed lysozyme samples were prepared in fetal bovine serum (FBS), human saliva and human urine. It was found that FAM emission was still visible upon addition of each mixed sample, implying that this assay has a great potential for the detection of real biological samples. This study illuminates that introduction of specific aptamer/protein interaction as the recognition event, and utilization of FRET as the signal transduction channel, is an effective way to develop CPE-based protein sensors with good specificity. 

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. 

In a similar fashion, aptamer-protein interactions can be monitored using intrinsic DNA and protein oxidation signals on carbon electrodes. The simultaneous monitoring of two signals from aptamer and protein enables us to detect the specific binding event in a rapid and easy format, as illustrated in Figure 7.1. 

Figure 7.1 Label-free voltammetric detection of aptamer-protein interactions. (A) The electrochemical oxidation response from the guanine bases in the aptamer is observed at about 1 V (peak G) on the surface of the screen-printed electrodes. (The inset shows gold and carbon-based screen-printed electrodes with a three-electrode system.) (B) After the binding event with the target protein, an oxidation signal appears at about 0.6 V (peak P), and the oxidation signal of the aptamer decreases.

In this mode redox probes to produce detectable signals are no longer covalently labeled onto the aptamers in this mode. Probes dissolved in electrolyte solution can interact with aptamers immobilized on an electrode via (1) electrostatic repel-lence (for negatively charged probes), (2) electrostatic adsorption (for positively charged probes), and (3) intercalation (for DNA intercalators). 

In the two studies using a "signal-off" approach by Xiao et al. [44, 45], thrombin was detected by monitoring the decrease in the amperometric response of a redox label present at one end of the thrombin aptamer as a result of the association of thrombin with the aptamer. The interaction of the labeled aptamer with its target modulates the distance of the electroactive labels from the sensor electrode, thereby altering the redox current. In the absence of the... 

The aptamer was modified with thiol at the 5 -end and biotin at the 3 -end, and both aptamer and HRP were immobilized on the AuNPs (GCE-Apt-AuNPs-HRP). After the aptamer-MUCl interaction, the aptamer was disrupted and the biotin exposed was captured by the streptavidin-modified electrode. This signal-on" aptasensor exhibited good linear dynamic ranges (8.8-353.3 nM) and a low detection limit (2.2nM) for MUCl in PBS buffer. 

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