Immobilization hybridization

Figure 8 Chemiluminescent (A and B) and bioluminescent (C) detections for immobilized hybridizations of PCR product. Dg, digoxigenin Bt, biotin Ad, avidin. Procedure A [30] Biotin moiety is incorporated into PCR products during the amplification reaction, using one 5 -biotinylated primer. The product is hybridized with a Dg-labeled probe and is immobilized on streptavidin-coated magnetic beads. This capture reaction is carried out for 30 min at 37°C. A permanent magnet is used to sediment the beads during washing to remove unbound DNA. By incubation with the washed beads for 45 min at 37°C, anti-Dg antibody conjugated to HRP enzyme is bound to the Dg-labeled probe, and luminol reaction is performed for CL detection. Procedure B [31] Wells of apolystyrene microtiter plate are activated with l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and then coated with a labeled cDNA probe complementary to an internal region of the target DNA.

The immobilized-hybridization methods provide simple detection systems that do not require separation of the target PCR product from contaminating DNA. These protocols are also applicable to the detection of an unmodified DNA target [5, 33, 34],... 

To elucidate the intramolecular interaction within the hybrid, CaM-PDE hybrid with EDC was immobilized onto DEAE-Sapharose, because immobilized hybrids were expected to have no interaction with each other (inter-hybrid interaction). The CaM-PDE hybrid was immobilized to DEAE-Sepharose suspended in 100 mM glycylglycine buffer (pH 7.5). In the presence of Ca2+, the CaM-PDE hybrid showed the activity change in its bound form. The activity of the CaM-PDE hybrid in the absence of Ca2+ was ca.40% as compared to that in the presence of Ca2+. The normalized modulation in the immobilized form is comparable to that in the free form, although the total modulated activity was smaller in the DEAE-Sepharose immobilized form as presented in Fig.30. These results indicate that the selfmodulation of CaM-PDE activity can be performed in its immobilized form, as this modulation was caused by the CaM moiety in the hybrid (intrainteraction). If CaM and PDE are independently present in solution, CaM has to randomly access to PDE to modulate the enzyme. However, in the case of CaM-PDE distinct CaM molecules modulates the corresponding PDE molecules in intra-hybrid interaction. In other words, CaM concentration... 

Compared to genosensors based on GEC, the novelty of this approach is in part attributed to the simplicity of its design, combining the hybridization and the immobilization of DNA in one analytical step. The optimum time for the one-step immobilization/hybridization procedure was found to be 60 min [66]. The proposed DNA biosensor design has proven to be successful in using a simple bulk modification step, hence, overcoming the complicated pre-treatment steps associated with other DNA biosensor designs. Additionally, the use of a one-step immobilization and hybridization procedure reduces the experimental time. Stability studies conducted demonstrate the capability of the same electrode to be used for a 12-week period [66]. 

The hybridization event can be detected both with label-free or enzymatic labelling procedures. The single-point attachment of DNA can be achieved by the immobilization of biotinylated DNA on Av-GEB platform. In this case, a one-step immobilization/hybridization procedure is achieved. The capability of surface regeneration of the biocomposite electrodes allows repeated analyses with the same electrode as... 

When the electrochemical detection is based on enz3mriatic activity determination, a capture format was used in which the immobilization of the biotinylated probe together with the hybridization was performed in a one-step procedure. The procedure consists briefly of the following steps, as schematically outlined in Fig. 3.5 (i) one-step immobilization/hybridization procedure... 

Figure 14.3. Schematic representation of the immobilization, hybridization, and detection of probe DNA.

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