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80�C0.82 Abs OD600 nm (5 �� 109 CFU/mL) bacteria cell concentration at 2 min response time and room temperature. In the meantime, taking the biosensor fluorescence signal once a week over a period of ten weeks helped us assess the biosensor��s lifetime. The bacterial cell membranes were kept at 4 ��C in the fridge when they were not in use. 2.8. Evaluation of Single and Combined Metal Toxicities via Biosensor and Microtox Assay The biosensor inhibition response was investigated by using a single toxicant [Cu(II), Cd(II), Pb(II), Zn(II), Cr(VI), Co(II), Ni(II), Ag(I) AZD3965 supplier and Fe(III)] and toxic cocktail [Cu(II), Pb(II) and Zn(II)]. The immobilized bacterial cell concentration was held constant at 5 �� 109 CFU/mL. The fluorescence inhibition signal from S6 Kinase the immobilized GFP E coli was monitored at fluorescence excitation and emission wavelengths of 400 �� 2 nm and 485 �� 2 nm, respectively. The biosensor fluorescence response was recorded before and after exposure to toxicants. The toxicity levels of single toxicants and toxicant mixture were calculated using Equations (1) and (2), respectively. %RFU=Fluorescence?intensity?with?analyteFluorescence?intensity?without?analyte?��100% (1) ��TU=A.?EC50%mixtureA.?EC50%single+B.?EC50%mixtureB.?EC50%single (2) where A and B are different metal concentrations coexisting in the toxicity mixture. EC50 is the effective concentration of a metal at 50% of its relative fluorescence unit (RFU). The EC50 parameters were evaluated by using probit regression model of Finney et al. [26]. This was estimated from linear regression parameters (probit equation, y = ax + b) at 95% confidence level with y and x as the observed probit and dose level of each test respectively. The values of a and b are the slope of the regression line and its intercept. The total toxicity unit of (��TU) = 1 indicates zero interaction 5-HT Receptor inhibitor additive effect between toxicants in a mixture, whilst ��TU >1 indicates an antagonistic effect, and its additive index (AI) value can be calculated with AI = [(?1) �� ��TU + 1]. ��TU