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Abstract
Abstract Complex water effluents pose a toxicity risk to biological wastewater treatments and environmental discharge. Dynamic sampling and risk assessment of effluents would mitigate downstream hazards, but few methods are available to assess microorganism toxicity on the minute timescale. To rapidly evaluate unknown aqueous effluents, a segmented-flow microfluidic device is refined for real-time gross toxicity detection and quantification. A microfluidic chip integrating a magnetic stirrer for serial dilution is demonstrated to rapidly determine EC50 values of known and unknown toxicants, where 100 nL samples are suspended into a continuous oil phase with no interfering surfactants. A five logarithmic dilution sequence is evaluated in zinc > copper > nickel cations. Resazurin concentration of 10 μM is optimal for Enterococcus faecalis at 0.1 optical density. The adaptable method is transferable to other microorganisms, such as common baker's yeast, Saccharomyces cerevisiae, where the resazurin reduction rate is 30% of Enterococcus faecalis (4 nM/s vs. 13 nM/s per 100 nL droplet). Zinc and nickel cations are observed to increase the base resazurin reduction rate of baker's yeast by 25%, whereas copper is found to be more cytotoxic than mercury cations.
Highlights
Water toxicity monitoring relies on manual sampling and lengthy protocols [1]
Segmented-flow microfluidic technologies have allowed traditional laboratory assays to be performed with high throughput and minimal reagent consumption [4], including antibiotics assessment based on resazurin to resorufin metabolism [5] or on E. coli genetically engineered to produce green fluorescent protein [6]
Droplets containing serial dilutions of E. faecalis harvested in the stationary phase, 0 or 6 mg L−1 Cu2+ and 10 μM resazurin are generated to assess the effect of bacterial density on the apparent toxicity of copper
Summary
Water toxicity monitoring relies on manual sampling and lengthy protocols [1]. Automated real-time toxicity monitoring would be an ideal alternative, as it could allow continuous water monitoring against bioterrorism, enforcing discharge regulations, and dynamic feedback in waste water treatment plants. Segmented-flow microfluidic technologies have allowed traditional laboratory assays to be performed with high throughput and minimal reagent consumption [4], including antibiotics assessment based on resazurin to resorufin metabolism [5] or on E. coli genetically engineered to produce green fluorescent protein [6] These high-throughput assays are focused on sample throughput with little to no focus on sampling, optimizing sample incubation time, or providing feedback to real-time operations. The platform is further expanded to generate logarithmic concentration gradients in < 3 min, designed with components for future portability (i.e. pressure-driven, magnetic stirring) These advantages are leveraged to explore the specificities of the resazurin reduction assay when performed in microfluidic format under continuous operation. To demonstrate the versatility of this microfluidic platform across microorganisms, bacteria were substituted with Baker's yeast that may be employed as a sacrificial bioreporter due to its wide availability, long storage capabilities (> 2 years dry) and minimal culture requirements
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