Thrombin aptamer immobilization

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). 

Wang Y, Liu B (2009) Conjugated polyelectrolyte sensitized fluorescent detection of thrombin in blood serum using aptamer-immobilized silica nanoparticles as the platform. Langmuir 25 12787-12793... 

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. 

Figure 5.2 SPR sensor response as a fnnction of thrombin concentration for different types of aptamer immobilization and different types of aptamer APTA immobilized on a (1) streptavidin layer (2) avidin (3) dendrimers covered by avidin (4) SH-APTA. For comparison, nonspecific interaction of HSA with a sensor snrface created by APTA immobilized on a streptavidin layer is shown (cnrve 5). The composition of aptamers was as follows APTA 3 -biotin-GGG TTT TCA CTT TTG TGG GTT GGT GTG GTT GG-5, SH-APTA SH-(CH2)e-GGT TGG TGT GGT TGG-5. Results represent meaniS.D. obtained from three independent experiments. [Adapted from Ostatnd et al. (2008), with permission of Springer.]...

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... 

Figure 8.1 Amplified detection of thrombin in solntion (A) and on snrfaces (B) by the catalytic enlargement of gold nanoparticles. The assay has a sandwich-like format and is based on the capture of thrombin by immobilized aptamer. A gold nanoparticle-labeled secondary aptamer is then added. Finally, the gold nanopartlcles are enlarged in a solution containing HAuCU and CTAB as a surfactant in the presence of NADH. [From Pavlov et al. (2004).]...

A mixed self-assembled monolayer was used for the aptamer immobilization on the gold electrode. The aptamer-modified electrodes were then incubated for 1 h at 37°C with thrombin (18 ag/mL). Electrochemical measurements were recorded in the thin-layer cell configured to contain a total volume of 20 [xL. Thrombin chromogenic substrate (/3-Ala-Gly-Arg-p-nitroaniline) was injected into the cell and differential pulse voltammetry (DPV) measurements between -0.2 and -1V with a pulse height of—0.05 V and pulse duration of 70 ms were carried out. The DPV measurements showed that /3-Ala-Gly-Arg-p-nitroaniline substrate and the p-nitroaniline product have different redox potentials. Moreover, the DPV experiments showed a current peak at -0.45 V in the presence of the thrombin substrate. After 5 min, the peak at —0.45 V decreased and a new peak was detected at -0.70 V, indicating the formation of p-nitroaniline. The same measurements carried out on a control electrode in order to test the specificity of the assay in this experiment bovine serum albumin (BSA) substituted thrombin and in this case only the peak at 0.45 V was measured. 

In another work [49], the thrombin aptamer was hybridized with a ferrocene-labeled DNA oligonucleotide and immobilized onto a gold electrode. The binding of thrombin to the aptamer causes the displacement of the complementary oligonucleotide resulting in a decrease of current recorded at the electrode by differential pulse voltammetry (DPV). A linear range for the detection of thrombin between 0 and 10 nM was obtained. 

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). 

Different QDs have different voltammelric signals, the signal intensity corresponds to the DNA concentration, and the detection limit is 270 pM. QDs can also be used in the multianalyte electrochemical aptamer sensor. For example, CdS QD labeled thrombin and PbS QD labeled lysozyme were captured by aptamers immobilized on a gold substrate. After addition of target proteins, QDs can be displaced and detected by the electrochemical method. This method is sensitive and selective, allowing simultaneous quantification of picomolar levels of proteins. 

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. 

For the experiment, the SWNT-FETs were prepared using standard chemical vapor deposition technique. Aptamer immobilization was performed by first modifying the sidewall of the carbon nanotube with CDI-Tween. While the Tween component was bound to the carbon nanotube sidewall through hydrophobic interactions, the carbodii-midazole (GDI) moiety was used to covalently attach the S amine group of the thrombin aptamer. Then, the devices were allowed to react with a (100 pM) thrombin... 

Another method for the analysis of aptamer-protein complexes involved the use of a positively charged ferrocene-tethered polythiophene, (19), as redox label reporting unit (Fig. 12.19). The antithrombin aptamer was immobilized on an electrode surface, and the electrostatic binding of the redox polymer (19) to the aptamer monolayer resulted in a supramolecular complex that revealed electrical contact between the polymer and the electrode.74 The formation of the aptamer-thrombin complex removed the polymer from the surface and blocked the electrical contact between the polymer label and the electrode. As a result, higher concentrations of thrombin increased the surface coverage of the aptamer-thrombin complex on the electrode, and this decreased the amperometric responses of the sensing device. 

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