ABSTRACT
We report the synthesis and the characterisation of the first electroactive ferrocene-labelled oligonucleotide phosphorothioate (ODN-Fc-Ps) probe obtained by automated synthesis. The grafting of ODN-Fc-Ps probe on gold electrode resulted in the appearance of the ferrocene redox couple in cyclic voltammetry confirming the effectiveness of the ODN grafting. The electrochemical response of the modified electrode was analysed in aqueous media before and after hybridisation with ODN target. The hybridisation with ODN target induces a large conformational change in the surface-confined DNA structure monitored by cyclic voltammetry of the ferrocene marker which confirms the potential of ferrocene-labelled oligonucleotide phosphorothioate to develop electrochemical DNA chips.
Subject(s)
DNA Probes/chemistry , DNA/chemistry , Ferrous Compounds/chemistry , Oligonucleotide Array Sequence Analysis/instrumentation , Oligonucleotide Array Sequence Analysis/methods , Robotics/methods , Thionucleotides/chemistry , Equipment Design , Equipment Failure Analysis , Metallocenes , Staining and Labeling/methodsABSTRACT
A new solution-phase phosphoramidite approach is reported for oligonucleotide synthesis employing recyclable solid-supported reagents. It uses polyvinyl pyridinium tosylate as the activator of a nucleoside-3'-O-phosphoramidite in the coupling step with a 5'-OH nucleoside or dinucleotide. The resulting phosphite triester was either sulfurized or oxidized using polystyrene-bound trimethylammonium tetrathionate or periodiate. This method avoids complicated purification steps, as excess reagents are easily removed by filtration. [reaction: see text]
Subject(s)
Oligonucleotides/chemical synthesis , Polymers/chemistry , Catalysis , Indicators and Reagents , Molecular Structure , Oligonucleotides/chemistryABSTRACT
We have developed new ferrocenyl-modified oligonucleotide (ODN) probes for electrochemical DNA sensors. A monofunctional ferrocene containing phosphoramidite group has been prepared, and a new bisfunctional ferrocene containing phosphoramidite and dimethoxytrityl (DMT) groups has been developed. These ferrocenyl-phosphoramidites have been directly employed in an automated solid-phase DNA synthesizer using phosphoramidite chemistry. The advantages of this method are that it allows a non-specialist in nucleotide chemistry to access labeled ODNs and that it has demonstrated good results. ODNs modified at the 3' and/or 5' extremities have been prepared, with the incorporation of the ferrocenyl group into the chain. The 5' position appears to be more important due to its particular behavior. The thermal stability and electrochemical properties of these new ODN ferrocenes were analyzed before and after hybridization with different ODNs. The feasibility of using these new ferrocenyl-labeled ODNs in DNA sensors has been demonstrated.
Subject(s)
Biosensing Techniques/methods , DNA/analysis , Ferrous Compounds/chemistry , Nucleic Acid Hybridization/methods , Oligonucleotide Probes/chemistry , Organophosphorus Compounds/chemistry , Electrochemistry , Metallocenes , Nucleic Acid Denaturation , Oligonucleotide Probes/chemical synthesisABSTRACT
The use of 1-[3-O-(2-cyanoethyl-N,N-diisopropylphosphor amidityl)propyl]ferrocene and 1-[3-O-dimethoxytrityl propyl]-1'-[3'-O-(2-cyanoethyl-N,N-diisopropylphosphoramidityl) propyl] ferrocene as reactive synthons for DNA/RNA synthesizer allows to generate ferrocene-labelled oligonucleotides with remarkable DNA detection properties.
