WP1. Design, synthesis and characterization of redox labeled NTPs and ddNTPs
Due to their tunable redox properties, high thermal stability and diversity, we have first selected polyoxometalates (POMs) as ideal candidates to offer the expected redox active compounds with distinct redox potentials. Various POMs have been synthesized first by varying the heteroatoms (functionalized Keggin type POMs SiW11O39SnC2H4COOH, PW11O39SnC2H4COOH, AlW11O39SnC2H4COOH, GeW11O39SnC2H4COOH), then by replacing two of the W with Mo to get PMo2W9O39SnC2H4COOH. In addition, we work with the Dawson type POM P2W17O61SnC2H4COOH.
Among the POMs synthesized we selected SiW11O39SnC2H4COOH (Keggin) and P2W17O61SnC2H4COOH (Dawson) to couple them with DNA primer, dNTPs and ddNTPs. First, we have optimized the method to conjugate the POMswith DNA primers. The POM-DNA conjugates were characterized by ESI MS, gel electrophoresis and electrochemical techniques. Then, we proved the compatibility of POMs with the functioning of polymerases in PCR reaction and the stability of the POM-DNA conjugates under those conditions. The results are published Chem. Eur. J. (2015) 21, 17721–17727. We have also tried to couple the above-mentioned POMs with the four DNA bases (dNTPs) through amide bonds. ESI-MS analysis proved that purine bases are coupled efficiently with POMs, while the pyrimidine bases are showing low signal in the ESI MS spectra. We are preparing part of this work for publication in high impact factor journal.
We are now preparing precursors for copper free click reaction with POMs and pyrimidine bases as alternative coupling strategy. We already experienced that POM DNA coupling using the copper mediated click reaction didn’t work.The use of POM modified purine nucleotides in PCR is under optimization.
We are also checking other redox active compounds in addition to POMs to find effective redox labels for the intended purpose. Ferrocene, phenothiazine, anthraquinone and methylene blue have already been utilized for labeling ddNTPs and dNTPs (part of the results are published in Chem. Comm. (2016) 52, 757-759). The latter three have been observed to suffer from some unwanted interaction with DNA and this hinders them from further use in isothermal DNA amplification using recombinase polymerase at 37°C. Ferrocene modified dNTPs have been comfortably used in similar amplification.
WP2. Electrode fabrication, optimization and characterization novel surface chemistries
As mentioned in the summary, the development of thermostable surface anchoring of ssDNA is highly crucial to achieve reliable and reproducible results during the diagnosis process in the final device. Literature survey on the various surface chemistries reported shows either loss of immobilized probes at high temperature or difficulty in the preparation of probe immobilized surfaces. The usually used thiolated molecules for surface functionalization are reported to be stable only up to 60°C. In our work we opted to develop a novel probe surface tethering strategy. Inspired by our linkage between POMs and organic side chains via tin anchors, we are optimizing novel tin chemistry on carbon and gold surfaces. The products are characterized by XPS, AFM, PM-IRRAS, SEM, and various electrochemical techniques.Preliminary observations show a highly thermostable self-assembled layer on gold electrodes. The GCE surface seems to contain pinholes, although the oxidation and reduction currents of ferrocyanide have considerably decreased. we are preparing manuscript to publish part of the work > Ahmed M. Debela, Mayreli Ortiz, Serge Thorimbert, Ciara K. O´Sullivan and Bernold Hasenknopf , versatile tool for fabrication of robust surface chemistries utilizing trichlorostananes .(under preparation)
WP3. PCR and Primer extension feasibility studies with labeled primers as well as dNTPs
This is partly mentioned WP1, and we will continue to evaluate the various POM labeled DNAs in PCR and PEX reactions.
WP4. Design and engineering of simple, robust and reliable fluidic system
The work planned for year 2
WP5. Evaluation of novel E-detection schemes for the detection of selected genetic disease
The work planned for year 2
