Handheld Fluorescent Lysosomal Yeast (FLY) Biosensor for Toxic Metal Ions andOrganic Pollutants in Water

About This Grant

Summary Abstract Nanohmics proposes to further develop its already proven general water toxicity biosensor based on the published principle that yeast cell vacuoles respond like human lysosomes by acidifying, swelling and increasing in numbers in response to a variety of pollutants in water within 30-60 minutes. Since lysosomes can be rapidly stained with a variety of pH-sensitive and lipophilic fluorescent dyes, monitoring lysosomal or vacuolar fluorescence levels can be used as a general indicator of water toxicity. Dried yeast can last for years in sealed envelopes across a large range of temperatures. Thus, the proposed Fluorescent Lysosomal Yeast (“FLY”) biosensor that uses dried Saccharomyces cerevisiae or other yeast would be extremely robust and exhibit long shelf life. Under a recently completed successful DoD Phase I SBIR, the FLY system was proven effective for detecting lethal levels of Hg (II), azide, nicotine, and phenol in water in addition to various other toxins and metal ions that Nguyen et al. have detected previously and reported using a similar FLY-based system. In Phase I, Nanohmics will investigate the ability of Saccharomyces yeast to respond to Cd II, Cr VI, Cu II, and Pb II, diazinon, malathion, glyphosate, PFAS compounds, and TCE at low to lethal levels. Yeast will be suctioned down onto filters in microwells in 96-well plates or 8- well strips for staining, washing, and fluorescence imaging in a benchtop apparatus in Phase I and a rugged, inexpensive (< $100) ~ 2 lb. handheld, battery-operated FLY biosensor prototype will be constructed for tap and well water testing. Nanohmics will also enhance the sensitivity and detection speed of FLY by adding a photomultiplier tube (PMT) for greater fluorescence signal to noise ratios and pre-concentrating analytes with hydrophobic matrices and chelators in portable columns, then desorbing the pollutants and metal ions in water samples at higher concentrations prior to introduction to the FLY biosensor. Finally, Nanohmics will investigate the feasibility of “Aptagate” approaches to specific analyte detection such as transfecting yeast with plasmids containing DNA aptamers as toxicant-binding transcriptional regulatory elements to either block RNA polymerase and turn off GFP synthesis and fluorescence (Iyer & Dokcytz FASEB J. 2012) or use RNA aptamers linked to ribozymes in Mckeague’s pDIVE (DNA Integrated Versatile Encoded) aptamer approach (Chem. Sci. 15:4770. 2024) for “lights on” GFP fluorescence detection as a function of toxicant concentration within 60 minutes.