Microwell fluoride assay screening for enzymatic defluorination

Area of Science:

• Environmental Science
• Biochemistry
• Analytical Chemistry

Background:

• Fluorinated compounds, including per- and polyfluorinated alkyl substances (PFAS), are persistent environmental pollutants.
• Microbial enzymes are key to environmental remediation of fluorinated compounds.
• Accurate fluoride determination is essential for monitoring defluorinating enzyme activity.

Purpose of the Study:

• To develop a rapid, sensitive, and high-throughput method for fluoride determination in aqueous samples.
• To facilitate the discovery and engineering of novel defluorinating enzymes for environmental cleanup.
• To improve upon existing low-throughput methods like ion chromatography and fluoride electrodes.

Main Methods:

• Adapted a lanthanum alizarin complexone-based drinking water test for enzyme and cell culture assays.
• Implemented the assay in a microtiter well plate format.
• Utilized color imaging and spectrophotometric plate reading for detection.

Main Results:

• Achieved detection of as little as 4 nmol of fluoride in 200 μL of assay buffer.
• Demonstrated amenability to automated liquid handling and high-throughput screening.
• Established a method suitable for analyzing thousands of enzymes and/or substrates.

Conclusions:

• The refashioned microtiter plate assay offers a significant improvement in speed and sensitivity for fluoride detection.
• This method will accelerate the identification and optimization of enzymes for defluorination of persistent organic pollutants.
• Enables efficient screening for microbial solutions to environmental fluorinated compound contamination.