Microwell fluoride assay screening for enzymatic defluorination

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Scopus citations

Abstract

There is intense interest in removing fluorinated compounds from the environment, environments are most efficiently remediated by microbial enzymes, and defluorinating enzymes are readily monitored by fluoride determination. Fluorine is the most electronegative element. Consequently, all mechanisms of enzymatic C-F bond cleavage produce fluoride anion, F. Therefore, methods for the determination of fluoride are critical for C-F enzymology and apply to any fluorinated organic compounds, including PFAS, or per- and polyfluorinated alkyl substances. The biodegradation of most PFAS chemicals is rare or unknown. Accordingly, identifying new enzymes, or re-engineering the known defluorinases, will require rapid and sensitive methods for measuring fluoride in aqueous media. Most studies currently use ion chromatography or fluoride specific electrodes which are relatively sensitive but low throughput. The methods here describe refashioning a drinking water test to efficiently determine fluoride in enzyme and cell culture reaction mixtures. The method is based on lanthanum alizarin complexone binding of fluoride. Reworking the method to a microtiter well plate format allows detection of as little as 4 nmol of fluoride in 200 μL of assay buffer. The method is amenable to color imaging, spectrophotometric plate reading and automated liquid handling to expedite assays with thousands of enzymes and/or substrates for discovering and improving enzymatic defluorination.

Original languageEnglish (US)
Title of host publicationFluorine Metabolism, Transport and Enzymatic Chemistry
EditorsRandy B. Stockbridge
PublisherAcademic Press Inc.
Pages65-83
Number of pages19
ISBN (Print)9780443236433
DOIs
StatePublished - Jan 2024

Publication series

NameMethods in Enzymology
Volume696
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Bibliographical note

Publisher Copyright:
© 2024

Keywords

  • Assay
  • Bacteria
  • Defluorination
  • Enzyme
  • Fluoride
  • High throughput
  • Microwell
  • Organofluorine
  • PFAS

PubMed: MeSH publication types

  • Journal Article

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