Immobilization DNA

Construction of the DNA chip (Nicolini 1996) requires a single-helix DNA immobilized at the surface of the device and sensitive to the binding of the complementary strand. 

The initial hurdle to overcome in the biosensor application of a nucleic acid is that involving its stable attachment on a transducing element which commonly includes a metallic electrode. In the first part of this chapter, we wish to introduce our approach for DNA immobilization (Scheme 1). A detailed characterization of the immobilization chemistry is also presented. In the second part, we follow the development of work from our laboratory on chemical sensor applications of the DNA-modified electrode involving a biosensor for DNA-binding molecules and an electrochemical gene sensor. 

FIG. 7 The quinacrine-dependent changes in cyclic voltammograms of the DNA-immobilized An electrode. Experimental conditions are the same as those in Fig. 5. 

Yokoyama K, Taira S (2005) Self-Assembly DNA-Conjugated Polymer for DNA Immobilization on Chip. 262 91-112 Yoshikawa I, see Araki K (2005) 256 133-165... 

Generally, there are a number of ways in which the adsorption and binding of charged macromolecules (in particular, DNA immobilization and hybridization) can affect the electrochemical properties of the analyte-FED interface. In the case of field-effect devices, two basic effects are usually considered ... 

The preparation of the FEDs, the experimental set-up and measuring conditions for the detection of DNA immobilization and hybridization as well as for the monitoring of the layer-by-layer adsorption of the polyelectrolyte multilayers are described in detail elsewhere [46-50], The attachment of these charged macromolecules to the FED surfaces has been systematically characterized by means of capacitance-voltage,... 

At the same time, however, it must be concluded that the practical development of FEDs for a label-free detection of DNA and other charged macromolecules by their intrinsic molecular charge seems to be more complicated than originally expected. Although the discussed results are highly exciting, they are rather diverse and even sometimes inconsistent. Factors influencing the DNA immobilization and hybridization detection by FEDs are ... 

F. Caruso, E. Rodda, D.F. Furlong, K. Niikura, and Y. Okahata, Quartz crystal microbalance study of DNA immobilization and hybridization for nucleic acid sensor development. Anal. Chem. 69, 2043-2049 (1997). 

DNA and tripropylamine. Alternatively, target DNA immobilized on the film could be determined by hybridization with a Ru(bpy)32+-labeled complementary DNA strand. 

Surface plasmon resonance studies were employed to measure the equilibrium constants and association and dissociation rate constants of bisnaphthalimide derivatives (20, 21) with hairpin DNA immobilized on the metal surface.123 The equilibrium constants were higher and the dynamics slower for compounds 20 and 21 when compared to the equilibrium constants and dynamics of the model monomer (19). The values for ka and kd were determined from the change in the surface plasmon resonance signal when, respectively, the ligand solution was flowed over the... 

The effect of structural modification to 22 on the binding efficiency and binding dynamics of hairpin DNA immobilized on metal surfaces were studied using surface plasmon resonance.124,125 The association and dissociation kinetics were analyzed using a sequential model for the binding of the imidazole containing polyamides... 

QDs recognized specific antigens/antibodies -DNA immobilization to QDs surfaces and possibility of hybrid assemblies [35] -Coupled to transferrin, QDs underwent receptor-mediated endocytosis in cultured HeLa cells... 

If a single-stranded DNA molecule is placed with a complementary single DNA sequence the two molecules will hybridize. This hybridization forms the basis of a number of very powerful techniques for detecting and quantifying specific nucleic acid sequences. The hybridization may be carried out either in solution or more commonly with the DNA immobilized on nitrocellulose filters. The complementary DNA sequence is known as a cDNA probe. Probes for a large number of important nucleic acid sequences are now available. 

Fig. 6 Common silanizing trialkoxysilanes that promote covalent DNA immobilization a 3-aminopropyltriethoxysilane (APTES) b 3-aminopropyltrimethoxysilane (APTMS) c 3-glycidoxypropyltrimethoxysUane (GOPS) d 3-mercaptopropyltrimethoxysilane (MPTS)...

Keywords DNA immobilization Surface chemistry Surface activation Patterning ... 

A truly systematic and integrated study is required to compare and contrast, as well as optimize the approaches described in this chapter for DNA immobilization to provide an industry standard for the future. 

Keywords DNA diagnostics Impedimetric detection DNA hybridization DNA immobilization... 

Examples of the various methods of DNA immobilization are shown in Fig. 5. Amongst these are physical adsorption and covalent immobilization. The covalent immobihzation is best exemplified by the use of organo-silane chemistries and alkanethiol chemistries. 

Fig. 5 Depictions of DNA immobilization by a covalent immobilization and b physical adsorption...

Fig. 6 Common silanizing agents to promote covalent DNA immobilization a 3-amino-propyltrimethoxysilane (APS) b 3-mercaptopropyltrime1hoxysilane (MPS) c 3-glycidoxy-propyltrimethoxysilane (GPS)...

DNA immobilized by organosilane chemistries has proven to be an effective method for measuring impedance changes upon hybridization using both gold and platinum electrodes [21,50]. The effect of different silane chemistries creates differences in the hydrophobicity and hydration levels of the modified surface. The organosilane treatment along with the ssDNA... 

Flowever, the focus of the major part of the chapters lies on the couphng chemistry used for DNA immobilization. Successful immobihzation techniques for DNA appear to either involve a multi-site attachment of DNA (preferentially by electrochemical and/or physical adsorption) or a single-point attachment of DNA (mainly by surface activation and covalent immobihzation or (strept)avidin-biotin linkage). Immobilization methods described here comprise physical or electrochemical adsorption, cross-linking or entrapment in polymeric films, (strept)avidin-biotin complexation, a surface activation via self-assembled monolayers using thiol linker chemistry or silanization procedures, and finally covalent coupling strategies. 

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