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CD-MB: Development of an electrochemical Molecular Beacon based DNA Chip for Coeliac disease predisposition diagnosis

Final Report Summary - CD-MB (CD-MB: Development of an electrochemical Molecular Beacon based DNA Chip for Coeliac disease predisposition diagnosis)

The aim of the CD-MB project was to develop a reagent-less/wash-less electrochemical sensor for the fast HLA typing of genes associate to the coeliac disease. This was considered important because HLA typing has been shown to be a valuable tool in the diagnosis of gene associate disease as coeliac disease or diabetes.

The fast and reagent less detection of the targeted genetic marker was envisaged to be achievable by the combination of nano-structured electrochemical devices and electrochemical molecular beacon (E-MB). Molecular beacons are synthetic molecular probes, based on short oligonucleotides chain, characterised by a peculiar stem-loop structure; this structure offers to the E-MB various advantages as:

i) high specificity;
ii) the possibility to carry an inherent signal transduction mechanism;
iii) and the possibility of detecting the target in a rapid and simply way (no need for wash or reagents).
Furthermore E-MB can be easily implemented in low-cost miniaturised / portable devices.

The use of nano-structured surface, via the deposition / assembling of nano-particles was seen to increase the density of the E-MB onto the sensor surface and, as a result, to improve the sensor sensitivity.

Project summary:

At the beginning of the project, three main tasks were identified: the definition of the required probes, the optimisation of the nano-structuring of the surface and the integration of the different elements in a sensing platform.

First step in the development of the envisaged sensor was the definition of the alleles that were required to be detected and to design the associated probes.

A total of six alleles was identified: DQA1*0501, DQA1*0505, DQB1*0201, DQB1*0302, DQA1*0201 and DQA1*0301;with the last two alleles not proven to be responsible for the coeliac disease but strongly correlated to the other alleles in positive patients. In order to specifically detect the different alleles, a total of seven probes were designed. Screening of the specificity of the different probes was performed using techniques as enzyme linked oligonucleotide assay (ELONA) or surface plasmon resonance (SPR). The results of these experiments showed that the probes designed were suitable for the selective detection of the targeted alleles.

Conversion of the proposed probes into E-MB was made possible by the introduction, at each end of the tested probes, of five bases reverse complementary one to the other; these chains allowed the closing of the different MB probes in their stem and loop structure.

In order to provide the electrochemical property to the E-MBs, the introduction of a methylene blue moiety was introduced at one of the end of the oligonucleotides probes. The choice of methylene blue was consistent with previous finding of the fellow that showed an higher stability of this redox label if compared with the other commercially available (Ferrocene). Finally, a grafting molecule, in this case a thiol, was introduced to the second end of the E-MB to allow their immobilisation onto the sensor surface.

We defined the design of the recognition elements, the following step for the definition of the approaches for the nano-structuring of the sensor surface. Two different approaches were identified and investigated. In the first approach the sensor surface was modified using a sequence of electrodeposition of the nano-particles and self assembling of the MB. This approach allowed the formation of an high density of Mb onto the sensor surface.

Aim was the preparation of surface presenting more homogeneous distribution of the nano-particles, both spatially and in size, the electro-spraying of solution containing Au nano-particle was performed. When electro-spraying was performed using Au nano-particle (80 nm) having size comparable with those of the drop obtained from the jet a quite uniform distribution of them onto the surface was obtained.

Conclusion:


In the CD-MB project, a full set of E-MB probes suitable for the low to medium resolution HLA typing of the coeliac disease associated genes was defined. Furthermore, the possibility of using electro-spray as a way of producing regular nano patterning of surfaces was explored and demonstrated.

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