Diagnostic area
DEVELOPMENT OF AN ELECTROCHEMICAL BIOSENSOR FOR DETECTION OF DNA MUTATIONS
Developing strategies for detection of specific DNA sequences is of great interest for the diagnosis of diseases caused by genetic alterations. Molecular diagnostics based on analysis of short DNA sequences provides sensitive and quantitative methods for early detection of these diseases. DNA hybridization is the most common process to analyze and detect a particular gene or a segment of a nucleic acid. Among the hybridization-based methods are those based on surface plasmon resonance, fluorescence, gravimetry or labeling with radioisotopes, and in recent years has had a boom in the development of DNA biosensors. Among these biosensors, the electrochemical methods are presented as very sensitive, allowing also develop portable, simple and inexpensive devices for clinical diagnostic studies.
Since DNA has electroactive properties, electrochemical systems have been developed for detection by taking advantage of this feature to directly detect the hybridized DNA. In general, DNA is immobilized on an electrode, and the difference in electrical current measured before and after hybridization is related to the amount of DNA fixed to the electrode. However, this untraced direct detection is not very selective. In order to improve the selectivity and sensitivity, different approaches have been developed using an electroactive marker. There are different electroactive compounds that may be associated with DNA and thus be detected by using an oxidation-reduction potential to the electrode. As these markers have better electroactive redox properties of DNA, its use allows to obtain a higher signal /noise ratio and higher sensitivity. The interaction of these electroactive compounds with DNA can be electrostatic (low specificity), being placed in the grooves of the double helix (weak bond) or intercalative process (union more specific and strong).
This project has developed a new molecule as an indicator of hybridization in electrochemical biosensors, prepared "in situ", and formed by the junction between the Pentamino complex ruthenium (Ru) and ligand 3 - (2-fenantren-9-ilvinil) pyridine (L). It has been called Pentamino ruthenium complex [3 - (2-fenantren-9-yl-vinyl) pyridine] “Rul”.
This complex has a dual role. On the one hand, the planar structure of the ligand aromatic groups of 3 - (2-fenantren-9-yl-vinyl) pyridine (L) gives intercalative character to the complex and allows its to join at the double-stranded nucleic acids in a very specific away. On the other hand, the redox center of the metal (Ru) is used as an electrochemical indicator to detect the hybridization event.
The DNA electrochemical biosensors developed in this project consist of a probe of single-stranded DNA, derivatized at its 5 'end with an alquiltiol, which allowing the chemisorption of the probe to the electrode. This gives a monolayer of DNA molecules organized on the surface of the electrode. In the optimal experimental conditions, this probe specifically reacts with complementary DNA strands. After that, the accumulation of electrochemical indicator RuL is made by applying a sequence of cyclic potential sweeps. The RuL is intercalated only where hybridization happend at a rate of approximately one molecule RuL for each four base pairs. The last step is to transform the process of hybridization in an electrochemical signal, applying an electrical potential electrode that oxidizes the ruthenium redox center. This oxidation leads to an electric current which is measured by Differential Pulse Voltammetry (DPV). The measurements are as the following graphics:
Figure 1. Differential pulse voltammogram (DPVs) of RuL after hybridization with a fully complementary sequence (WT), after hybridization with a sequence in a single mismatched base (MUT), and before hybridization (WT -SH). The WT-SH signal is similar to the one obtained with hybridization with a not complementary sequence.
The biosensor’s device in this project is an electrochemical cell in which three electrodes are integrated: a gold working electrode (WE) which is attached the DNA probe, a platinum counter electrode (CE), and a silver reference electrode (RE).
The high sensitivity and specificity achieved with this method, through the use of RuL complex as an indicator of hybridization, allow the detection of a particular sequence of DNA and its quantification. Also, allows the detection of a mismatched base in a sequence of DNA double helix and the position of the mismatch within the sequence. All this gives to the electrochemical biosensor a great potential for application in clinical diagnosis.