Alizarin Red S for Online Pyrophosphate Detection Identified by a Rapid Screening Method.
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.
Division Molecular Biotechnology and Functional Genomics, Technical University of Applied Sciences Wildau, Hochschulring 1, 15745 Wildau, Germany.
Jens
J
Fischbach
Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Penang, Malaysia.
Qiuting
Q
Loh
Institute for Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Straße 24-25, 14476 Potsdam, Germany.
Frank F
FF
Bier
Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Penang, Malaysia.|Analytical Biochemistry Research Centre, Universiti Sains Malaysia, 11800 Penang, Malaysia.
Theam Soon
TS
Lim
Division Molecular Biotechnology and Functional Genomics, Technical University of Applied Sciences Wildau, Hochschulring 1, 15745 Wildau, Germany.
Marcus
M
Frohme
Division Molecular Biotechnology and Functional Genomics, Technical University of Applied Sciences Wildau, Hochschulring 1, 15745 Wildau, Germany.
Jörn
J
Glökler