Periodic Reporting for period 1 - [e-POM-Bioanal] (Electrobioanalytical Applications of Polyoxometalates) Reporting period: 2015-06-01 to 2017-05-31 Summary of the context and overall objectives of the project In this project we are developing a novel diagnostic tool for easy and accurate detection of disease causing genes by employing electrochemical methods. To develop this tool we have to identify four different types of redox active compounds with distinct redox potential between –0.8V and 0.8V. In addition to this, the redox labels should be compatible with DNA-polymerases, and stable under physiological and PCR working condition. The other important issue is the development of robust,thermostable surface chemistry for DNA grafting, which can withstand the elevated temperature used during enzymatic incorporation. If the above-mentioned critical steps are achieved, the work remaining will be the detection of selected genetic diseases using our electrochemical diagnostic tool and validate the tool with real samples. The diagnostic devise will allow for highly cost-effective, rapid and user-friendly platforms for the screening of known and unknown genetic mutations and known single nucleotide polymorphisms. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far WP1. Design, synthesis and characterization of redox labeled NTPs and ddNTPsDue 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 chemistriesAs 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 dNTPsThis 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 systemThe work planned for year 2WP5. Evaluation of novel E-detection schemes for the detection of selected genetic disease The work planned for year 2 Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Update of the plan for exploitation and dissemination of result (if applicable)The researcher has been located for his first year in the group of Prof. Serge Thorimbert. Since May 2016 all the IPCM members moved into new refurbished buildings, and he is now directly integrated in the group of Prof. B. Hasenknopf. He participates in the weekly scientific seminars every Monday for general chemistry, and every Tuesday for group meetings. The researcher was also trained in the use of different equipment and techniques such as: HPLC, electrochemistry, and various laboratory techniques. The researcher has participated in the following conferences and workshops: • Training session: European Patent Academy Workshop AW17-2015; Intellectual property rights workshop for Marie Skłodowska-Curie Fellows, 24-25 November 2015, Munich, Germany.• Training session: Advanced analytical strategy using Electrochemical Impedence Spectroscopy at Laboratory of Interfaces and electrochemical systems (LISE) – UPMC, Paris. 6 days between 06/04/ and 09/05/2016. • Poster presentation: International symposium on surface chemistry beyond self-assembly toward functional covalent materials, organized by observatory for micro and nanotechnology (OMNT) on May 10th 2016, Paris.So far the researcher has published two papers and is preparing two more for submission to high impact factor journals: • Ahmed M. Debela, Serge Thorimbert, Mayreli Ortiz, Bernold Hasenknopf and Ciara K. O´Sullivan, Electrochemical primer extension for the detection of single nucleotide polymorphisms in the cardiomyopathy associated MYH7 gene, Chemical Communications 52 (4), 757-759 (I.F 6.9)• Ahmed M. Debela, Mayreli Ortiz, Valerio Beni, Serge Thorimbert, Denis Lesag, Cole Richared, Ciara K. O´Sullivan, Bernold Hasenknopf, Polyoxometalate biofunctionalised primers: Use in polymerase chain reaction and electrochemical detection of PCR product, Chemistry–A European Journal 21 (49), 17721-17727 (I.F 5.931) preparation-of-pom-dntps.gif
