Nucleic acid probe, immobilization

An array or a matrix of nucleic acid probes immobilized at discrete locations on a silicon or glass surface provides a convenient means to simultaneously probe a sample for the presence of many different target sequences. Microarray biochip scanning devices, mostly based on fluorescent labels, are now currently available, and could also be used with CL labels to take advantage of the higher sensitivity of this detection principle. 

Figure 1. (a) Recognition of a solution-borne nucleic acid (target) by a nucleic acid probe immobilized on a surface, (b) Fluorescence microscope image of a 1.28 cm X 1.28 cm probe array with 200 pm x 200 pm synthesis sites on which labeled target oligonucleotides have adsorbed. 

A Brief Practical Example for the Immobilization of Nucleic Acid Probes... 

An array of oligonucleotide which is composed of 20 80-mer oligos, or peptide nucleic acid probes, is synthesized either in situ (i.e., on-chip) or using conventional synthesis followed by on-chip immobilization. The resultant DNA array is then exposed to the labeled sample of DNA, hybridized, and the identity/abundance of complementary sequences determined. This method was developed at Affymetrix, Inc. and called DNA chips. Today, oligonucleotide-based chips are manufactured by many companies using alternative in situ synthesis or depositioning technologies. 

Several useful schemes for attaching nucleic acid probes onto electrode surfaces have thus been developed [2-8]. The exact immobilization protocol often depends on the electrode material used for signal transduction. Common probe immobilization schemes include attachment of biotin-functionalized probes to avidin-coated surfaces [15], self-assembly of organized monolayers of thiol-functionalized probes onto gold transducers [16], carbodiimide covalent binding to an activated surface [17], as well as adsorptive accumulation onto carbon-paste or thick-film carbon electrodes [15-30]. 

Another work reported the immobilization of nueleie aeids on CNTs previously opened by oxidative treatment and proteeted by a spin-on-glass film [69]. Once the carboxyl residues were obtained they were activated with EDC and NHS and the nucleic acids were immobilized (Scheme 3). The hybridization event in this case was evaluated by using a peptide nucleic acid sequence as probe and a target containing a fluorescent residue. 

The immobilization of nucleic acid probes is also important. Various schemes can be used for attaching the DNA probes to the surface. These include the use of thiolated DNA for self-assembly onto gold surfaces, covalent... 

Solid phase hybridization is in most cases achieved on membranes. Target nucleic acid is immobilized and subsequently detected by a probe. This approach forms the basis of slot/dot blot hybridization, Northern and Southern hybridization and colony or plaque hybridization. Dot/slot blot hybridization (Kafatos et al., 1979) demonstrates the presence of target sequences but not their size. Although solid phase hybridization is convenient for hybrid/free probe separation, it has the disadvantages that nucleic acid is most often bound noncovalently and that targets are immobilized at fre-... 

Sandwich hybridization, using affinity-based hybrid collection, is based on two nonoverlapping nucleic acid probes (one is labeled, the other can be collected by the affinity matrix) (Syvanen et al., 1986 Jalava et al., 1990). The principles are shown in Fig. 8.3. Target nucleic acid thus mediates binding of labeled probe to the matrix. The detectability is about 10 molecules with a linear range to at least 10 molecules with radioisotopes as labels. In contrast to capture hybridization assays, the immobilization of the complex is at 22-37°C (leaching is then usually less important). 

Will mixed biorecognition units be used, for example, antibodies and nucleic acid probes This complicates the system but very often may offer far greater analytical potential. A key issue that must be addressed is how the biorecognition unit will be immobilized to optimize its performance [139]. 

The immobilization of the nucleic acid probe is crucial in determining the performance of the biosensor. To achieve high sensitivity and selectivity, the hybridization efficiency must be maximized and the non-specific adsorption minimized. Immobilization should produce a stable layer of well-defined probe orientation, readily accessible to the target. There are a wide variety of immobilization methods, depending on the transducer surface and application. For... 

In addition, electrocatalytic detection of surface-immobilized nucleic acid probes using the Ru(bpy)3 + mediator is highly dependent upon the preparation of ITO surface [321]. To maximize electron-transfer between Ru(bpy)3 + and ITO, the charge at ITO needs to be negative. Phos-phonated SAM was allowed to form on ITO by immersing the electrode in solution of 1 mM 12-phosphonododecanoic acid in 50% DMSO/50% H2O for 16.5 h. ITO electrodes were removed from solution and washed with successive 10 min rinses of H20,1 M NaCl, and H2O [321]. 

Abel and co-workers [106] at Ciba-Geigy Ltd. have reported an automated optical biosensor system. Their device utilizes 5 -biotinylated-16-mer oligonucleotide probes bound to an optical fiber functionalised with avidin to detect complementary oligonucleotides pre-labeled with fluorescein moieties in a total internal reflection fluorescence (TIRF) evanescent wave motif similar to that of Squirrell. Immobilization of nucleic acid probes onto the optical fiber substrate was achieved by functionalisation of the surface with (3-aminopropyl)triethoxysilane (APTES) or mercaptomethyldimethylethoxysilane (MDS). Onto the short alkylsilane layer was... 

Biological/Medical Applications Characterizing cyclodextrin-based polyrotaxanes detecting nucleic acids, - polyamines immobilizing proteins " probing lysozyme in human serum as a substrate for measuring polymerases activity," recombinases activity," topoisomerases activity" ... 

Nucleic acid hybridization methods use oligonucleotide DNA probes with sequences complementary to a portion of the nucleic acid of the target bacterium38,60 and designed to hybridize with immobilized DNA or RNA on a membrane. After any unbound probe has been washed off, the hybridized probe can be detected.64 66... 

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