Solid-phase hybridization assay

When the nucleic acid target or probe is immobilized on a solid support, the kinetics of hybridization are even more complex. Many of the preceding observations stfil hold true, but the rate and extent of solid-phase hybridization are lower than with solution-phase hybridization. Depending on the concentrations of the reactants, solid-phase hybridization can be either nucleation-hmited or diffusion-limited. Optimal efficiency of solid-phase hybridization is achieved under conditions that facilitate diffusion of the probe to the support and that favor hybridization over strand-reassociation if double-stranded probes are used. This usually means a small volume of hybridization solution and relatively low probe concentrations. In practice, solid-phase hybridization assays are empirically designed. Time of hybridization and probe concentration are the two variables most frequently adjusted in the assay. Conditions that tend to maximize the extent of hybridization and minimize the background or nonspecific attachment of the probe are selected. 

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. 

The main difference between the capture and the sandwich assays is how the label probe-target complex is immobilized on a solid phase. In capture assays, hybridization with the immobilized capture probe determines the kinetics and specificity/detectability/sensitivity characteristics of the assay. In sandwich assays, an immobilized molecule, which is not a nucleic acid (e.g., streptavidin, antibodies), serves to capture the hapten probe-target-label probe ternary complex (thus both probes need to be modified). The affinity matrix-hapten interaction thus determines the kinetics (usually 3-10 times faster than solid phase hybridization), the sensitivity and detectability... 

Fig. 7.4 Examples of solid-phase ECL assay formats, a DNA hyinidization assay based on an immobilized ssDNA hybridizes with a labeled target ssDNA. b Sandwieh-type DNA biosensor, c Assay used for integrase aetivity test with immobilized and Iree-labeled dsDNA. d Sandwich-type immunoassay, e Direct immunoassay, f Competitive assay in which analyte competes with labeled analyte for antibody-binding sites on immobilized antibody, g Protease activity assay in which cleavage of the immobilized peptide results in the decrease in ECL emission due to the removal of the ECL label, h Kinase activity assay using a labeled antibody to recognize the phosphorylated product. Adapted from Ref. [23]. Copyright 2008 American Chemical Society...

Other recombinant studies showed that recombinant N-terminal and other regions of fibrillin-1 have a tendency to dimerise (Ashworth et al, 1999b Trask et al., 1999). An unpaired cysteine residue in the first hybrid domain, which contains nine cysteines, is predicted to be involved in covalently linking N-terminally-aligned fibrillin-1 molecules (Reinhardt et al., 2000b). N- and C-terminal halves of the fibrillin-1 molecule, and the N-terminal half of fibrillin-2, were shown to interact with high affinity in solid-phase and BIAcore binding assays (Tiedemann et al., 2001). However, N- and C-terminal fibrillin-2 polypeptides did not interact with each other. These data demonstrated that fibrillins can direcdy interact in an N- to C-terminal fashion to form homotypic fibrillin-1 or heterotypic fibrillin-1/fibrillin-2 microfibrils. 

Nakagami et al. (1991) proposed a different universal probe system. In their procedure, phagemids are used to obtain strand-specific ss DNA molecules, one with the insert complementary to the target sequence and a second without the insert and with the opposite polarity (Fig. 7.23). The ss DNA without insert is labeled with any label (usually a secondary label) and this labeled DNA is hybridized with the complementary strand containing the insert. This leaves only the probe sequence ss whereas the tag sequence is ds. This method has several advantages (i) like the sandwich method it leaves the probe sequence single-stranded (ii) hybridization of the tag sequences in solution instead of on the solid phase as in a sandwich assay is much faster (iii) no fragmentation is required. 

Immunoassays for nucleic acids usually employ a solid phase on which the analyte is immobilized either before or after hybridization. Two general configurations, immunocapture or hybridization capture (enzyme-linked oligonucleotide-sorbent assay or ELOSA), are possible (Figure 1). Careful design and optimization of each component can yield a high-performance assay. Typical components for a microwell PCR immunoassay are provided in Table 1. 

The most significant limitation of immunochemical methods is inadequate sensitivity such that preamplification by PCR is required. Further improvements in solid phases, reporter enzymes, substrates, and hybridization probes will extend the range of analytes amenable to assay without amplification. Solid phases designed specifically for DNA immunoassays are needed. Enzyme engineering for thermostability. 

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