Design and Development of Fluoride Riboswitch Based Biosensor to Detect the Fluoride Level in Drinking Water

Abstract

Fluoride plays a major role in human health as it is categorized as an essential micronutrient. Fluoride
may cause adverse effects or beneficial effects depending on the concentration present in the human
body. Since drinking water plays an important role in the fluoride diet, it is crucial to determine the
fluoride concentration in the drinking water to reduce the vulnerability of communities to fluoride -
dependent diseases. However, there are many drawbacks associated with the conventional methods
for fluoride detection, such as interference with other anions, which greatly limits the scope of their
use. Bacterial riboswitches are well known for their high selectivity towards the ligand. The detection
method utilizes an Escherichia coli mutant carrying a plasmid cloned with fluoride responsive
riboswitch, coupled to the lacZ reporter gene. LacZ, which is under the regulation of fluorideriboswitch, encodes β-galactosidase in response to the intracellular fluoride levels. The activity of βgalactosidase was determined by the turn-over of its substrate ONPG, which forms a yellow color
product ONP which absorbs light at 420 nm. The ONP absorbance was used to quantify the fluoride
level in the aqueous samples. The calibration curve showed a linear relationship (R2=0.9842) for the
concentrations between 5-100 µM. Next, the biosensor was used to measure the groundwater samples
collected from different regions of Sri Lanka. The highest level of groundwater fluoride was found
in Ampara, Batticaloa, Kurunegala (>2 ppm), and the lowest level was found in Colombo and Matara
(<0.5 ppm). The experimentally determined fluoride levels are in agreement with the reported values.
Our results also demonstrate that the novel biosensor is highly selective for fluoride and can readily
discriminate between chloride and hydroxyl ions. This work provides a critical proof-of-principle for
utilizing the fluoride riboswitch-based whole-cell biosensor to selectively and accurately detect
fluoride levels in drinking water.

Keywords: Fluoride, Riboswitch, LacZ