Genosensors hybridization

Moreover, the unique adsorption properties of GEC allowed the very sensitive electrochemical detection of DNA based on its intrinsic oxidation signal that was shown to be strongly dependent of the multi-site attachment of DNA and the proximity of G residues to GEC [100]. The thick layer of DNA adsorbed on GEC was more accessible for hybridization than those in nylon membranes obtained with genosensors based on nylon/GEC with a changeable membrane [99,101,102]. Allhough GEC has a rough surface, it is impermeable, while nylon is more porous and permeable. DNA assays made on an impermeable support are less complex from a theoretical standpoint [7] the kinetics of the interactions are not compUcated by the diffusion of solvent and solutes into and out of pores or by multiple interactions that can occur once the DNA has entered a pore. This explained the lower hybridization time, the low nonspecific adsorplion and the low quantity of DNA adsorbed onto GEC compared to nylon membranes. 

Physical or electrochemical adsorption uses non-covalent forces to affix the nucleic acid to the solid support and represents a relatively simple mechanism for attachment that is easy to automate. Adsorption was favoured and described in some chapters as suitable immobilization technique when multisite attachment of DNA is needed to exploit the intrinsic DNA oxidation signal in hybridization reactions. Dendrimers such as polyamidoamine with a high density of terminal amino groups have been reported to increase the surface coverage of physically adsorbed DNA to the surface. Furthermore, electrochemical adsorption is described as a useful immobihzation strategy for electrochemical genosensor fabrication. 

A disposable electrochemical enzyme-amplified genosensor was described for specific detection of Salmonella (Del Giallo et al., 2005). A DNA probe specific for Salmonella was immobilized onto screen-printed carbon electrodes and allowed to hybridize with a biotinylated PCR-amplified product of Salmonella. The hybridization reaction was detected using streptavidin conjugated-AP where the enzyme catalyzed the conversion of electroinactive a-naphthyl phosphate to electroactive a-naphthol, which was detected by differential pulse voltammetry. 

A genosensor, or gene-based biosensor/DNA biosensor, normally employs immobilized DNA probes as the recognition element and measures specific binding processes such as the formation of DNA-DNA and DNA-RNA hybrids, and the interactions between proteins or ligand molecules and DNA at the sensor surface [5]. 

The genosensor design based on Av-GEB not only is able to successfully immobilize onto the electrode surface the mecA biotin-labelled capture probe, while the hybridization with the mecA target and the mecA digoxigenin-labelled probe is occurring at the same time, but also is capable of distinguishing SNPs [58,65]. 

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]. 

Keywords Carbon screen printed electrodes Genosensors Electrochemical adsorption Hybridization event Electroactive indicators Indicator-free electrochemical genosensor... 

Wide-scale genetic testing requires the development of easy to use, fast, inexpensive, and miniaturized analytical devices. Hybridization DNA biosensors (also called genosensors) offer a promising alternative to traditional methods based on either direct sequencing or DNA hybridization, commonly too slow and labor intensive. 

Typically, the basic steps in the design of an electrochemical genosensor are (a) immobilization of the DNA probe, (b) hybridization with the target... 

In this section the different formats reported in literature of a genosensor based on electrochemical adsorption are described. The strategies reported in literature for the hybridization step and for the different procedures of detection of the hybrids will be summarized. Examples of applications to real samples are given. 

In literature two possibilities for the detection of a DNA hybridization event in the case of carbon SPE genosensor are reported to use electroactive indicators or to detect the guanine moiety signal per se (label-free). 

Sequence-Specific Hybridization Genosensors based on Electroactive Indicators... 

To give some examples, Co(bpy)33+ [19] or Co(phen)33+ [18,20-22] are associated with the surface hybrid by immersing the SPE in a solution containing the indicator and applying the potential of + 0.5 V vs. Ag/AgCl for 2 min. The surface-accumulated indicator is measured using CPSA at a constant current. The genosensor was used to detect DNA sequences related to the human immunodeficiency virus type l(HIV-l) [18], sequences related to Escherichia Coli [19], M. Tuberculosis [20], and Cryptosporidiumparvum [21],... 

Hybridization Biosensing based on the Guanine Signal (Indicator-Free Electrochemical Genosensor)... 

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. 

Many DNA-based biosensors (genosensors) are based on the ability of complementary nucleic acid strands to selectively form hybrid complexes. The complementary strands anneal to one another in a Watson-Crick manner of base pairing. Hybridization methods used today, such as microhtre plates or gel-based methods, are usually quite slow, requiring hours to days to produce reliable results, as described by Keller and Manak [10]. Biosensors offer a promising alternative for much faster hybridization assays. 

Among numerous reported applications of genosensors for DNA hybridization as few examples, one can refer to a disposable DNA sensor for detection of hepatitis B virus genome DNA,145 biosensor systems for homeland security using DNA microarrays,146 and DNA electrochemical biosensor with conducting polymer film and nanocomposite as matrices for detection of HIV DNA sequences.147... 

Matrix array hybridization on silicon wafers ( genosensors on chips )... 

Label-free electrochemical hybridization genosensor for the detection of hepatitis B virus genotype on the development of lamivudine resistance. Anal Chem 77, 4908-4917. 

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 ana-l5Aical step. 

Intercalated doxorubicin has been also used as an electrochemical label in the detection of DNA hybridization events in a genosensor built by layer-by-layer covalent attachment of multiwalled carbon nanotubes and Au-NPs [46]. The oxidation peak current obtained by differential pulse voltammetry showed a linear relationship with the logarithm of the target DNA concentration in the range 5.0 x 10 to 1.0 x 10 M, with a detection limit of 6.2 pM. 

Also, intercalated adriamycin has been used as hybridization label in a genosensor built by modifying a glassy carbon electrode (GCE) with multiwalled carbon nanotubes with carboxyl groups and Au-NPs [23], Differential pulse voltammetry (DPV) was utilized to monitor the DNA hybridization event. Under the optimal conditions, the increase of reduction peak current of adriamycin was linear with the logarithm of the concentration of the complementary oligonucleotides from 1.0 x 10 to 5.0 x 10 M with a detection limit of 3.5 x 10 M. 

DNA detection is usually performed by hybridization. For designing a genosensor, the crucial steps are the choice of the transducer surface and the immobilization of the single-stranded (ssDNA)... 

---