Alizarin Red S for Online Pyrophosphate Detection Identified by a Rapid Screening Method

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.

Isothermal amplifications – a comprehensive review on current methods

“…Thus, PPi formation in an isothermal amplification is indirectly detected by the depletion of free manganese ions (Tomita et al 2008). Other dyes such as Alizarin Red S (ARS) are also useful to detect PPi in combination with manganese (Fischbach, Loh, et al 2017). Accumulation of protons in the typical polymerase reaction can be detected by pH indicators such as Phenol red or Cresol red (Tanner et al 2015).…”

TbEu(BTC) as a dNTP label for LAMP outcome in nucleic acid testing

“…Phosphate sensors with reliable selectivity and high sensitivity can also give effective indirect measurement of amplification. There have been different examples of phosphate linked detection schemes [4], for example, Fischbach et al focused on the interaction of Alizarin Red S with the remaining free quencher ion (Mg 2 + and Mn 2 + ) [9]. Shi et.al.…”

“…There have been different examples of phosphate linked detection schemes [4], for example, Fischbach et al . focused on the interaction of Alizarin Red S with the remaining free quencher ion (Mg 2+ and Mn 2+ ) [9]. Shi et.al.…”

UV-Spectroscopic Detection of (Pyro-)Phosphate with the PUB Module

“…Despite their prevalence, the experimental interrogation of many of these enzyme functions (particularly their kinetics) remains difficult since the detection of inorganic (pyro-)phosphate is not trivial in many biochemical assays. Notable previous developments toward this end include fluorescent phosphate binding proteins, 2 fluorescent ionic dyes with tunable photochemical properties, 3 chromogenic metal complexes, 4 and coupled enzymatic assays employing purine nucleoside phosphorylases and xanthine oxidases to generate a UV signal. 5 However, these methods typically only allow for the examination of a small experimental space, exhibit low selectivity, or employ expensive reagents unsuited for high-throughput experimentation (see the Supporting Information and Table S1 for a detailed literature analysis).…”