Genosensors

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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. 

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. 

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. 

Del Giallo, M. L., Ariksoysal, D., Marrazza, G., Mascini, M., and Ozsoz, M. (2005). Disposable electrochemical enz5nne-amplified genosensor for Salmonella bacteria detection. Anal. Lett. 38, 2509-2523. 

Figure 20-19 Fiber-optic sensor lor detection of specific DNA sequences. Upper half shows circular wells in etched tip of bundle. Fluorescence image in lower half identifies wells to which fluorescent target DNA has bound. [From J R. Epstein. M. Lee. and D. R. Waft High-Density Fiber-Optic Genosensor Microsphere Array Capable of Zofriomoto Detection limits, ...

Chapters 1 to 5 deal with ionophore-based potentiometric sensors or ion-selective electrodes (ISEs). Chapters 6 to 11 cover voltammetric sensors and biosensors and their various applications. The third section (Chapter 12) is dedicated to gas analysis. Chapters 13 to 17 deal with enzyme based sensors. Chapters 18 to 22 are dedicated to immuno-sensors and genosensors. Chapters 23 to 29 cover thick and thin film based sensors and the final section (Chapters 30 to 38) is focused on novel trends in electrochemical sensor technologies based on electronic tongues, micro and nanotechnologies, nanomaterials, etc. 

Genosensor technology for electrochemical sensing of nucleic acids by using different transducers... 

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]. 

Typically, the design of a genosensor involves the following steps [7] (1) modification of the sensor surface to create an activated layer for the attachment of the DNA probe (2) immobilization of the probe... 

Recent developments in genosensor design with the advances in nanotechnology provide new tools in order to develop new techniques to monitor biorecognition and interaction events on solid surfaces and also in solution phase. Typical applications include environmental monitoring and control, and chemical measurements in the agriculture, food, and drug industries [8-17]. 

In aspect of chip-based technology, electrochemical genosensors based on different materials and transducers have been recently developed in response to clinical demand of giving promising results [18-25]. Different sensor technologies provide a unique platform in order to immobilize molecular receptors by adsorption, crosslinking or entrapment, complexation, covalent attachment, and other related methods on nanomaterials [5,7,26]. 

An overview on the genosensor technologies for detection of nucleic acids (NA) immobilized onto different transducers by adsorption, cross-linking, complexation and covalent attachment is briefly summarized in Table 19.1. The applications of electrochemical genosensor technology are discussed in the following section. 

In recent years, electrochemical genosensors developed on the principle of nanotechnology have become one of the most exciting forefront fields in analytical chemistry due to the recent advances in... 

M.I. Pividori, A. Merkoci and S. Alegret, Electrochemical genosensor design immobilisation of oligonucleotides onto transducer surfaces and detection methods, Biosens. Bioelectron., 15 (2000) 291-303. 

M. Yang, M.E. McGovern and M. Thompson, Genosensor technology and the detection of interfacial nucleic acid chemistry, Anal. Chim. Acta, 346 (1997) 259-275. 

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]. 

M.I. Pividori, A. Merko i, J. Barbe and S. Alegret, PCR-genosensor rapid test for detecting Salmonella, Electroanalysis, 15 (2003) 1815-1823. 

D. Hernandez-Santos, M. Diaz-Gonzalez, M.B. Gonzalez-Garcia and A. Costa-Garcia, Enzymatic genosensor on streptavidin-modified screen-printed carbon electrodes, Anal. Chem., 76 (2004) 6887-6893. 

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