@article{FischbachLohBieretal.2017, author = {Fischbach, Jens and Loh, Qiuting and Bier, Frank Fabian and Lim, Theam Soon and Frohme, Marcus and Gl{\"o}kler, J{\"o}rn}, title = {Alizarin Red S for Online Pyrophosphate Detection Identified by a Rapid Screening Method}, series = {Scientific Reports}, volume = {7}, journal = {Scientific Reports}, issn = {2045-2322}, doi = {10.1038/srep45085}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:526-opus4-9248}, year = {2017}, abstract = {We identified Alizarin Red S and other well known fluorescent dyes useful for the online detection of pyrophosphate in enzymatic assays, including the loop mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assays. An iterative screening was used for a selected set of compounds to first secure enzyme compatibility, evaluate inorganic pyrophosphate sensitivity in the presence of manganese as quencher and optimize conditions for an online detection. Of the selected dyes, the inexpensive alizarin red S was found to selectively detect pyrophosphate under LAMP and PCR conditions and is superior with respect to its defined red-shifted spectrum, long shelf life and low toxicity. In addition, the newly identified properties may also be useful in other enzymatic assays which do not generate nucleic acids but are based on inorganic pyrophosphate. Finally, we propose that our screening method may provide a blueprint for rapid screening of compounds for detecting inorganic pyrophosphate.}, language = {en} } @article{FischbachFrohmeGloekler2017, author = {Fischbach, Jens and Frohme, Marcus and Gl{\"o}kler, J{\"o}rn}, title = {Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) - A New Versatile Isothermal Amplification Method}, series = {Scientific Reports}, volume = {7}, journal = {Scientific Reports}, issn = {2045-2322}, doi = {10.1038/s41598-017-08067-x}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:526-opus4-9824}, year = {2017}, abstract = {The growing demand for cost-effective nucleic acid detection assays leads to an increasing number of different isothermal amplification reaction methods. However, all of the most efficient methods suffer from highly complex assay conditions due to the use of complicated primer sets and/or auxiliary enzymes. The present study describes the application of a new linker moiety that can be incorporated between a primer and a secondary target binding site which can act both as a block to polymerase extension as well as a hinge for refolding. This novel "hinge-primer" approach results in an efficient regeneration of the primer binding site and thus improves the strand-displacement and amplification process under isothermal conditions. Our investigations revealed that the reaction with forward and reverse hinge-primer including an abasic site is very efficient. The assay complexity can be reduced by combining the hinge-primer with a corresponding linear primer. Furthermore, the reaction speed can be increased by reducing the length of the amplified target sequence. We tested the sensitivity down to 104 copies and found a linear correlation between reaction time and input copy number. Our approach overcomes the usually cumbersome primer-design and extends the range of isothermal amplification methods using a polymerase with strand-displacement activity.}, language = {en} } @article{OmarLohTyeetal.2013, author = {Omar, Noorsharmimi and Loh, Qiuting and Tye, Gee Jun and Choong, Yee Siew and Noordin, Rahmah and Gl{\"o}kler, J{\"o}rn and Lim, Theam Soon}, title = {Development of an Antigen-DNAzyme Based Probe for a Direct Antibody-Antigen Assay Using the Intrinsic DNAzyme Activity of a Daunomycin Aptamer}, series = {Sensors}, volume = {14}, journal = {Sensors}, number = {1}, issn = {1424-8220}, doi = {10.3390/s140100346}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:526-opus4-6267}, pages = {346 -- 355}, year = {2013}, abstract = {G-Quadruplex (G-4) structures are formed when G-rich DNA sequences fold into intra- or intermolecular four-stranded structures in the presence of metal ions. G-4-hemin complexes are often effective peroxidase-mimicking DNAzymes that are applied in many detection systems. This work reports the application of a G-rich daunomycin-specific aptamer for the development of an antibody-antigen detection assay. We investigated the ability of the daunomycin aptamer to efficiently catalyze the hemin-dependent peroxidase activity independent of daunomycin. A reporter probe consisting of biotinylated antigen and daunomycin aptamer coupled to streptavidin gold nanoparticles was successfully used to generate a colorimetric readout. In conclusion, the daunomycin aptamer can function as a robust alternative DNAzyme for the development of colorimetric assays.}, language = {en} }