Detection of the Hybridization Event

The development of new transducing materials for DNA analysis is a key issue in the current research efforts of electrochemical-based DNA anal5Aical devices. While DNA immobilization and detection of the hybridization event are important features, the choice of a suitable electrochemical substrate is also of great importance in determining the overall performance of the anal3Aical electrochemical-based device, especially regarding the immobilization efficiency of DNA. 

The resulting CNTPE can also be modified by immersion, drop coating, or electrodeposition of different molecules, polymers, biomolecules, or metals. Some examples include 3,4-dihydroxybenzaldehyde [153] and polyOs-GDH-Dl-NAD" for the detection of glucose in alcoholic beverages [154] or pyrrole polymerized at CNTPE with a DNA probe for the detection of the hybridization event [155]. 

The background problem can be further overcome when using a surface-confined fluorescence excitation and detection scheme at a certain angle of incident light, total internal reflection (TIR) occurs at the interface of a dense (e.g. quartz) and less dense (e.g. water) medium. An evanescent wave is generated which penetrates into the less dense medium and decays exponentially. Optical detection of the binding event is restricted to the penetration depth of the evanescent field and thus to the surface-bound molecules. Fluorescence from unbound molecules in the bulk solution is not detected. In contrast to standard fluorescence scanners, which detect the fluorescence after hybridization, evanescent wave technology allows the measurement of real-time kinetics (www.zeptosens.com, www.affinity-sensors.com). 

The original colony hybridization method described the use of radio-labeled probes and the detection of positive hybridization events by autoradiography (1). However, because of the high waste disposal costs, short half-lives, long autoradiographic exposures, and potential health hazards associated with radioisotopes, there is interest in alternative methods to detect positive hybridizations. 

The electrochemical detection is an extensively used method to analyze specific DNA sequences by means of the hybridization event due to its simplicity, selectivity, low instrumentation costs, and high sensitivity. 

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. 

Electrochemical nucleic acid biosensors are based on electrochemical transduction of the hybridization event and show great promise for detection of specific gene sequences related to inherited and infectious diseases. Electrochemical detection of specific DNA sequences has an advantage in reducing the size of the total detection system [36], The advantages of electrochemical nucleic acid biosensors include potential of miniaturization, short response time, ease of use, low cost, and compatibility with microfabrication techniques [37],... 

In the following sections we will focus on the major steps involved in electrochemical DNA hybridization biosensors, namely the formation of the DNA recognition layer, the actual hybridization event, and the transformation of the hybridization event into an electrical signal (Figure 2). As will be illustrated below, the success of such devices requires a right combination of synthetic-organic and surface chemistries, DNA recognition, and electrochemical detection schemes. 

In contrast to the DNA detection by its intrinsic molecular charge, even in high ionic-strength solutions (0.5 M), where the hybridization efficiency is high and the hybridization event can be faster, a detectable sensor signal can be achieved. The estimations performed under the assumptions presented in Fig. 7.8 predict signal values of about 28-35 mV. 

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