Immobilization of DNA

Fiq. 60. Schematic illustrating electrostatic immobilization of DNA to a carboxyl-modified Si(lll) surface. Reproduced with permission from Ref. (194). Copyright 2000, American Chemical Society. 

Nucleic acids, DNA and RNA, are attractive biopolymers that can be used for biomedical applications [175,176], nanostructure fabrication [177,178], computing [179,180], and materials for electron-conduction [181,182]. Immobilization of DNA and RNA in well-defined nanostructures would be one of the most unique subjects in current nanotechnology. Unfortunately, a silica surface cannot usually adsorb duplex DNA in aqueous solution due to the electrostatic repulsion between the silica surface and polyanionic DNA. However, Fujiwara et al. recently found that duplex DNA in protonated phosphoric acid form can adsorb on mesoporous silicates, even in low-salt aqueous solution [183]. The DNA adsorption behavior depended much on the pore size of the mesoporous silica. Plausible models of DNA accommodation in mesopore silica channels are depicted in Figure 4.20. Inclusion of duplex DNA in mesoporous silicates with larger pores, around 3.8 nm diameter, would be accompanied by the formation of four water monolayers on the silica surface of the mesoporous inner channel (Figure 4.20A), where sufficient quantities of Si—OH groups remained after solvent extraction of the template (not by calcination). 

Y. Ishige, M. Shimoda, and M. Kamahori, Immobilization of DNA probes onto gold surface and its application to fully electric detection of DNA hybridization using field-effect transistor sensor. Jpn. J. Appl. Phys. 45, 3776-3783 (2006). 

Fig. 16.10 Reaction scheme for immobilization of DNA onto functionalized Si02 substrates. Plasma treated Si02 substrates are denoted as Si OH, APTMS functionalized substrates are denoted as Si NH2, dendrimer functionalized substrates are denoted as Si G(4.5)COOH, and substrates to which DNA capture probes have been immobilized are denoted as Si DNA. Inset Repeat unit of PAMAM dendrimer possessing terminal carboxylic acid functionality...

A very interesting approach was presented recently by Niemeyer et al. [113]. They prepared covalent DNA-streptavidin conjugates to which biotinylated alkaline phosphatase, beta-galactosidase, and horseradish peroxidase, as well as biotinylated anti-mouse and anti-rabbit immunoglobulins, were coupled. Immobilization of DNA-streptavidin conjugates was performed by hybridization with the complementary oligonucleotides, bound to the surface. It was demonstrated... 

Presented here is a concise description of surface immobilization of DNA, oligonucleotides, and DNA derivatives by adsorption onto carbonaceous materials, and the properties of the DNA layer adsorbed on carbonaceous solid phase. 

Compared with SWCNTs, the much cheaper MWCNTs produced by the CVD method are known to have more defects and can provide more sites for the immobilization of DNA. 

To take advantages of the unique properties of CNTs, a general approach is the immobilization of DNA on CNTs and the further immobilization of the DNA-modified CNTs on an easier-to-handle pure conductor, e.g., GC [26], Ft [121], Au [ 122]. Another approach consists of the prior modification of the pure conductor (GC) with the CNTs through dry-adsorption and the further DNA or DNA derivatives adsorption on the CNT-modified surface [123-125]. 

Electropolymerizable monomers that give rise to conducting electroactive polymers (CEPs) or insulating polymer thin films provide a convenient approach for the immobilization of DNA. More importantly, this method provides an easy means to achieve spatial separation of the ssDNA sequence... 

One crucial and hence central step in the design, fabrication and operation of DNA chips, DNA microarrays, genosensors and further DNA-based systems described here (e.g. nanometer-sized DNA crafted beads in microfluidic networks) is the immobilization of DNA on different soHd supports. Therefore, the main focus of these two volumes is on the immobilization chemistry, considering the various aspects of the immobihzation process itself, since different types of nucleic acids, support materials, surface activation chemistries and patterning tools are of key concern. 

Second, we tried the immobilization of DNA molecules as a potential target material for the immobilization of biological macromolecules. An aqueous solution of 1 mg/mL A.-DNA was spotted onto the surface of an azopolymer and covered with a cover glass, where the A.-DNA was stained with a fluorophore (YOYO-1 iodide, Molecular Probes Inc., Eugene, OR) in advance and the surface was then irradiated with the linearly shaped laser beam for 5 min, as shown in Fig. 3b. The surface was washed for 5 min in an aqueous solution and was then observed using... 

Several other methods are proposed, such as use of coulomb force between anionic DNA and cationic particle (7), use of biospecific affinity between biotin-labeled DNA and avidin-carrying particle (8), etc. Other methods for immobilization of DNA on the particle include reactions using cyanogen bromide, glutaraldehyde, and cyanuric chloride (9). 

Fig. 21.1. Schematic representation of the manipulation of DNA biosensors using different strategies. (A) DNA biosensors modified with DNA by (Al) dry-adsorption and (A2) wet-adsorption on GEC platform. (B) DNA biosensors based on the single-point immobilization of biotinylated DNA on Av-GEB universal affinity platform. (C) Immobilization of DNA on magnetic beads followed by (Cl) the capture of the modified beads on m-GEC electrode (more details in Pividori and Alegret [58]).

Compared to genosensors based on GEC, the novelty of this approach is in part attributed to the simplicity of its design, combining the hybridization and the immobilization of DNA in one analytical step. The optimum time for the one-step immobilization/hybridization procedure was found to be 60 min [66]. The proposed DNA biosensor design has proven to be successful in using a simple bulk modification step, hence, overcoming the complicated pre-treatment steps associated with other DNA biosensor designs. Additionally, the use of a one-step immobilization and hybridization procedure reduces the experimental time. Stability studies conducted demonstrate the capability of the same electrode to be used for a 12-week period [66]. 

K.M. Millan, A.L. Spurmanis and S.K. Mikkelsen, Covalent immobilization of DNA onto glassy carbon electrodes, Electroanalysis, 4 (1992) 929-932. 

S.R. Rasmussen, M.R. Larsen and S.E. Rasmussen, Covalent immobilization of DNA onto polystyrene microwells the molecules are only bound at the 5 end,Anal. Biochem., 198 (1991) 138-142. 

M.L Pividori and S. Alegret, DNA adsorption on carbonaceous materials. In C. Wittman (Ed.), Immobilization of DNA on Chips I, Topics in Current Chemistry, Vol. 260, Springer Verlag, Heidelberg, Berlin, 2005, pp. 1-36. 

Fig. 30.1. Schematic representation of the construction of GEC electrodes (steps (i)-(v)) and manipulation of the GEC electrodes, comprising the immobilization of DNA on GEC by dry-adsorption (vi) followed by incubation of the modified GEC electrode (vii).

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