Reverse target capture hybridization

As mentioned at the beginning of this review, covalent grafting of biomolecules onto colloidal support avoids reversible immobilization. Furthermore, an extended conformation of the ODNs is a key factor for efficiently hybridizing the complementary DNA fragment (i.e., target capturing). However, implementation of immobilization is more difficult than adsorption. 

Fig. 8.5. Reverse capture assays allow an improvement of detectability by a decrease in background staining. Two oligomers (labeled and polyadenylated) are hybridized with the target (I, II) and captured with paramagnetic beads (PMB) covered with oligo-dT hairs (III). Nonspecifically adsorbed oligomers are usually tighter bound than hybrids to dT-hairs, allowing a selective desorption of specific hybrids (IV). These can be recaptured (V), followed by the determination of the amount of label bound.

Assays based on sandwich-hybridization are available in several platforms, such as sequential injection analysis (55), microtiter plate assays (61), and microfluidic devices (62). The LFA biosensor assays described in this chapter rely on the sandwich-hybridization of a nucleic acid sequence based amplified (NASBA) RNA target between a membrane immobilized capture probe and SRB-encapsulating liposome conjugated reporter probe. NASBA uses the enzymes avian myeloblastosis virus reverse transcriptase (AMV-RT), RNaseH, and T7 DNA dependent RNA polymerase in the presence of deoxyribonucleoside triphosphates and appropriate primers to amplify relatively few copies of target RNA into... 

An alternative approach to the intrinsic DNA electrochemical activity utilizes electroactive species as redox indicators of the presence of immobilized DNA as well as its interaction events such as hybridization, damage, and association with another substance [14]. This mode was also used in a pioneering work on the DNA biosensor used for sequence detection [7]. In this case, it is still a label-free method in the sense that DNA probes or targets are not chemically modified by a special label however, as the indicator has to be added to a test S5 em as an additional reagent, we cannot speak more about the reagent-less technique. Redox indicators typically possess electrochemical responses at a "safe" electrode potential and often reversibly. The terms redox probe and redox marker are sometimes used in the literature to mean the redox indicator, which is confusable with the DNA capture probe used as a recognition element at hybridization and with markers used in medical diagnostics [8]. 

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