DIY enzyme labelled fluorescence alcohol (ELFA) standard production protocol to quantify single-cell phosphatase...

Extracellular enzyme activities (EEA) are crucial components of microbial food web interactions and biogeochemical cycles in aquatic ecosystems. They also represent relevant biological traits in the ecophysiology of phytoplankton and other components of microbial plankton. To assess species-specific and (sub-)population-level characteristics of phytoplankton EEA at the single-cell level and close-to-in-situ conditions solely the enzyme labelled fluorescence (ELF)-based substrates have been used, because they become fluorescent and precipitate around the enzyme activity location upon enzymatic cleavage. However, the enzyme-labelled fluorescence alcohol (ELFA) standard is no longer commercially available, hence standard curves cannot be run anymore and single-cell phosphatase activity (SCPA) is no longer quantifiable. Therefore, we introduce a simple protocol for an ELFA standard do it yourself (DIY) production to enable quantifying microplankton SCPA again. This protocol is based on fluorescence measurements easily available to environmental enzyme activity laboratories, and it circumvents any need for chemical synthesis equipment and knowledge. The method is based on a controlled reaction of the ELF-phosphate (ELFP) substrate with commercially available alkaline phosphatase, which efficiently turns all the substrate into ELFA product. The ELFA product was dried out and dissolved again in dimethyl sulfoxide (DMSO) for storage. The ELFA concentration of that standard-to-be ELFA solution in DMSO was determined by linear regression between a low concentration dilution series of ELFA solution measured fluorimetrically and parallel measurements of a series of phosphatase-catalysed reactions at an overlapping ELFP concentration range. Finally, the fluorescence- and concentration-stable ELFA solution in DMSO with a known concentration constitutes the ELFA standard that is necessary to quantify bulk (fluorimeter) and single-cell (microscope and flow cytometer) phosphatase activity in microplankton.