Immobilization of DNA

Thus, immobilizing DNA to a variety of materials is a key parameter to produce DNA biosensors. Materials supporting DNA sequences in biosensors are often called transducers. Currently, there are three general types of immobilization that are widely used: adsorption, avidin-biotin complexation, and covalent attachment (Pividori et al. 2000).

2.2.1 DNA Immobilization by Adsorption

Adsorption is the most convenient method among those three mentioned above to immobilize nucleic acids on transducer surfaces. It does not require reagents or special nucleic acid sequences, but the affinity between the DNA and transducer surface is necessary. Suitable materials for this type of immobilization include polystyrene, metal oxides (such as aluminum oxide, palladium oxide), nylon membranes, and carbon/carbon paste transducers (Wang et al. 1996a, b, c).

Electrochemical adsorption on carbon paste surfaces is one of the most common DNA immobilization methods. There are plenty of reports to modify either singlestrand DNA (ssDNA) or double-strand DNA (dsDNA) via a potential applied to carbon paste electrodes or to screen-printed carbon strip electrodes (Wang et al. 1996b, c; Wang et al. 1997a, b). For ssDNA, a voltage (vs. Ag/AgCl reference electrode) on a carbon transducer is applied for a couple minutes as a pretreatment. This pretreatment increases the roughness as well as hydrophilicity of the carbon surface (Wang et al. 1996a; Wang et al. 1997b). Afterwards, the electrochemical adsorption of ssDNA is implemented by stirring a solution on carbon surfaces at a potential for a preset time that depends on both the DNA sequence and concentration. The positive charge on carbon surfaces will enhance the stability of the immobilized DNA probe via electrostatic attraction. At the same time, such electrostatic attraction also increases the orientation of DNA probe alignment, so that a negative-charged hydrophilic sugar-phosphate backbone of the DNA probe can orient to carbon surfaces and the base part orients towards the solution readily hybridized with the target (Ronkainen et al. 2010; Palecek et al. 1998; Marrazza et al. 1999), as shown in Fig. 5. Hybridization will result in selective recognition and generation of biological signals for further diagnosis.

For dsDNA, no hybridization happens during the adsorption and detection process, so dsDNA electrochemical adsorption probes are suitable for non-DNA ana-lytes, which can bind to dsDNA; those include both electroactive and nonelectroactive analytes. Double-strand DNA probes are easier to mount on electrodes (less orientation requirements), and due to a lack of the need for hybridization, they usually have longer lifetimes than ssDNA probes (Wang et al. 1997b).

Besides electrochemical adsorption, physical adsorption is also used in DNA sensors. Briefly, the DNA biosensor is prepared by dipping a glassy carbon electrode into a dsDNA solution leaving the electrode to dry (Oliveira Brett et al. 1997, 1998).

DNA probe b

Complementary DNA (target)

Complementary DNA (target)

Electröchemical adsorption hybridization

Electröchemical adsorption hybridization

C2 Electroactive indicator (I)

Guanine (G) oxidation signal

Electroactive hybridization indicator (I)

Fig. 5 DNA immobilization by electrochemical adsorption. (a) DNA probe adsorption to an electrochemical transducer applying a positive potential. (b) Hybridization between the probe and the target, holding the same positive potential. (c) Transduction. (c1) Transduction using the guanine oxidation signal. (c2) Indicator preconcentration in the dsDNA at a fixed positive potential and transduction based on the electroactive hybridization indicator (adapted from Pividori et al.

C2 Electroactive indicator (I)

Guanine (G) oxidation signal

Electroactive hybridization indicator (I)

Fig. 5 DNA immobilization by electrochemical adsorption. (a) DNA probe adsorption to an electrochemical transducer applying a positive potential. (b) Hybridization between the probe and the target, holding the same positive potential. (c) Transduction. (c1) Transduction using the guanine oxidation signal. (c2) Indicator preconcentration in the dsDNA at a fixed positive potential and transduction based on the electroactive hybridization indicator (adapted from Pividori et al.

This sensor can be used in preconcentrating nitroimidazole or mitoxantrone on the surface and to study the interaction mechanism of these drug reactions with dsDNA by differential pulses, such as square wave voltammetry. Another method of physical dsDNA adsorption uses a glassy carbon disk. DNA-modified electrodes are prepared by the evaporation of a small volume of DNA solution on the electrode surface. The detection of the duplex was made with electroactive indicators (Cu(phen)22+ and Cu-(TAAB)2+) by cyclic voltammetric analysis of a preconcentrated electroactive indicator (Labuda et al. 1999).

Although adsorption is a simple and rapid method to immobilize DNA, the major disadvantage of this method is the labile connection between materials and DNA. As a result, DNA may dissociate from the transducer surface during hybridization conditions, causing low efficiency of hybridization. Thus, methods that rely on bond connections between DNA and transducer surface have been developed.

2.2.2 DNA Immobilization by Protein Complexation

Avidin or streptavidin-biotin complexation has been widely applied in the DNA biosensor field due to their high stability and high selectivity (Fig. 6). Avidin and

Fig. 6 DNA immobilization involving avidin-biotin complexation. (a) Avidin adsorption onto the graphite electrode. (b) Complexation between avidin and biotinylated DNA probe. (c) Hybridization of avidin-biotin probe with DNA target. (d) Signal transduction using an elec-troactive hybridization indicator preconcentrated into the dsDNA (adapted from Pividori et al. (2000))

Fig. 6 DNA immobilization involving avidin-biotin complexation. (a) Avidin adsorption onto the graphite electrode. (b) Complexation between avidin and biotinylated DNA probe. (c) Hybridization of avidin-biotin probe with DNA target. (d) Signal transduction using an elec-troactive hybridization indicator preconcentrated into the dsDNA (adapted from Pividori et al. (2000))

streptavidin are tetramer proteins (70 kDa) incorporating four identical binding sites. Biotin is a small molecule that specifically binds with avidin or streptavidin (Ka = 1 x 1015 L/mol). Thus, this binding effect has a similar energy gain to a cova-lent bond, so it is quite stable and can only be removed under the most extreme conditions. Such robust bonds make the complex unaffected even in extreme values of pH, relative high temperature, organic solvents, and denaturing agents.

The tetravalent binding of streptavidin to biotin allows a general procedure for constructing DNA-coated electrodes, where the surface-bound avidin is coupled to a biotinylated oligonucleotide. This is followed by an avidin treatment to block any adsorption of DNA on a material surface (or proteins that provide a biotin head outside). The inherent aqueous stability of the avidin-biotin complex renders the system easy to handle. Because of the presence of a large protein layer, there might be some nonspecific interactions to block DNA-DNA binding sites (Pividori et al. 2000; Marrazza et al. 1999). Although usually such blocking would not significantly impact detection, due to the extremely high selectivity of hybridization, a more desired and direct approach has been investigated for DNA immobilization: covalent bond connections.

2.2.3 DNA Immobilization by Covalent Bonds

Covalent bond attachment is a very stable immobilization modification on materials with usually simple structures and mechanisms. It can provide stable linkages which allows for increasing hybridization times. In addition, chemical linkers will provide more flexible structures that allow conformational change without losing nucleic acids.

In 1993, the first modification was developed to install a covalent bond onto a glassy carbon surface (Millan et al. 1992; Millan and Mikkelsen 1993). In this case, the electrode was electrochemically oxidized in an acidic solution to generate a carboxylic acid on carbon surfaces. After rinsing, carbodiimide 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide (EDC) and N-hydro-xysulfosuccinimide (NHS) were evaporated on the electrode surface. The activated electrode was then exposed to a small volume of ssDNA and was again allowed to dry. These steps allowed only a deoxyguanosine residue of ssDNA immobilized on the carbon surface (Fig. 7). Several years later, a similar modification method was developed to modify carbon paste electrodes in bulk by either octadecylamine or stearic acid (Mikkelsen 1996; Millan et al. 1994). Furthermore, researchers have developed an amino-DNA bond connection to modify graphite surfaces (Fig. 8) (Liu et al. 1996).

Besides carbon-based materials, DNA can be also immobilized onto inorganic surfaces (Hashimoto et al. 1994; Yang et al. 1997; Sun et al. 1998). For instance, because of the high affinity of gold to thioalcohol, researchers are trying to make thiol-nucleic acids self-assemble with gold surfaces. Self-assembled aminoethanethiol -modified gold nanoparticles have been successfully connected to ssDNA.

Fig. 7 Immobilization of ssDNA on glassy carbon electrodes

Eletrochemical /?

oxidation & '/ LiAlH3

APTES

EDC SS w w

Fig. 8 Immobilization of ssDNA on graphite surfaces by using an amino-DNA linkage (APTES 3-aminopropyltriethoxysilane)

Moreover, ssDNA is added after generating a thiol-derivative monolayer to achieve a more precise way to control the coverage of gold surfaces. Such methods greatly reduce nonspecific DNA adsorption (Herne and Tarlov 1997; Steel et al. 1998). For polymer transducers, this methodology has been used to prepare immobilized DNA probes electrochemically using direct copolymerization of pyrrole and oligonucle-otides bearing a pyrrole group (Livache et al. 1994). The process includes a direct electrochemical copolymerization of pyrrole and ssDNA with a pyrrole moiety introduced by phosphoramidate chemistry at their 5' end. Finally, a pyrrole group is covalently linked to a synthetic ssDNA.

In summary, adsorption is a fast, simple, and low-cost method to immobilize DNA onto biosensors. In contrast, covalent attachment usually needs more steps to build chemical bonds, but it is a highly tailorable method, which can connect with different materials. Therefore, depending on the specific application, a variety of methods are available for DNA immobilization.

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