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 <5 min, enabling feedback for dynamic processes e.g. dilution, ion exchange, or absorbant treatments. The resazurin-based assay is refined from traditional laboratory procedures to reduce sample volume and response time, with the advantages of short toxicant exposure (< 30 s), high density bacteria (10^7 cell/mL), and continuous mixing in an oxygen-free medium. Simple scaling of the stirred chamber volume from 1 to 30 μL adjusts the number of samples from 100 to 2500 dilution droplets, respectively. A detailed analysis of the resazurin kinetics suggests the presence of two or more enzymes with distinct Michaelis constants. Enzyme kinetics and the resazurin reduction rate is dependent on growth phase and EC50 inhibition by mercury > 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.
Ortiz, R., Chen, J. L., Stuckey, D. C., & Steele, T. W. J. (2019). Rapid serial diluting biomicrofluidic provides EC50 in minutes. Micro and Nano Engineering, 2, 92–103. https://doi.org/10.1016/j.mne.2019.02.002