Genosensors hybridization assay

Many DNA-based biosensors (genosensors) are based on the ability of complementary nucleic acid strands to selectively form hybrid complexes. The complementary strands anneal to one another in a Watson-Crick manner of base pairing. Hybridization methods used today, such as microhtre plates or gel-based methods, are usually quite slow, requiring hours to days to produce reliable results, as described by Keller and Manak [10]. Biosensors offer a promising alternative for much faster hybridization assays. 

Figure 9.4. Schematic representation of a impedimetric genosensor [sandwich hybridization assay]. Unmodified PGR products [b] were captured at the sensor interface [a] via sandwich hybridization with the surface-tethered probe and a biotinylated signaling probe. The biotinylated hybrid [c] was then coupled with a streptavidin-alkaline phosphatase conjugate [d] and finally exposed to the substrate solution [e]. The biocatalyzed precipitation of an insulating product [f] blocked the electrical communication between the gold surface and the [Fe(CN]6] redox probe [published by Elsevier in Ref. 50].

Moreover, the unique adsorption properties of GEC allowed the very sensitive electrochemical detection of DNA based on its intrinsic oxidation signal that was shown to be strongly dependent of the multi-site attachment of DNA and the proximity of G residues to GEC [100]. The thick layer of DNA adsorbed on GEC was more accessible for hybridization than those in nylon membranes obtained with genosensors based on nylon/GEC with a changeable membrane [99,101,102]. Allhough GEC has a rough surface, it is impermeable, while nylon is more porous and permeable. DNA assays made on an impermeable support are less complex from a theoretical standpoint [7] the kinetics of the interactions are not compUcated by the diffusion of solvent and solutes into and out of pores or by multiple interactions that can occur once the DNA has entered a pore. This explained the lower hybridization time, the low nonspecific adsorplion and the low quantity of DNA adsorbed onto GEC compared to nylon membranes. 

The development of assay techniques that have convenience of solid-phase hybridization and are rapid and sensitive will have a significant impact on diagnostics and genomics [3]. In this respect, SPE genosensors have several advantages they are safe because they are disposable, they are reproducible, they are inexpensive, and the overall procedure is quite fast. In this respect, electrochemical adsorption (adsorption controlled by a positive potential) is an easy to perform and rapid way of immobilization. The method does not require special reagents or nucleic acid modifications. 

Moreover, the hybridization reaction with noncomplementary target does not occur for all concentrations assayed (see the voltammogram in Fig. 9.13B for the highest concentration of noncomplementary target assayed, 700 fg/pL). This fact shows that non-specific adsorptions are not observed. Regarding the selectivity of the genosensor, this system has been studied in the previous section and this is able to discriminate one-base mismatched strands. 

The general scheme of a genosensor assay development starts with the immobilization of the specific nucleic acid sequence ("probe") on the transducer surface. The presence of the complementary sequence ("target") in the sample is recognized and captured by the probe through hybridization. 

Thus, in this assay, rapid method has eliminated the two steps that are normally included in the conventional electrochemical genosensor assay the denaturation of the PCR amplicon and its hybridization. Here, we merely immobilized the biotin- and fluorescein-labeled PCR amplicon on a streptavidin-modified SPC, followed by incubation with HRP-conjugated anti-fluorescein antibody, and the direct detection of the amperometric signal [8]. However, there is a need to incorporate PCR and electrochemical analysis into a single device for this method to be fully usable for field applications [13]. 

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