Label-Free Critical Micelle Concentration Determination of Bacterial Quorum Sensing Molecules  B.M. Davis, J.L. Richens, P. O'Shea  Biophysical Journal  Volume 101, Issue 1, Pages 245-254 (July 2011) DOI: 10.1016/j.bpj.2011.05.033 Copyright © 2011 Biophysical Society Terms and Conditions

Figure 1 Diagrammatic representation of the measurement of CMC using the method outlined in the text. Biophysical Journal 2011 101, 245-254DOI: (10.1016/j.bpj.2011.05.033) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 2 (A) Determination of oleic acid critical micelle concentration (CMC) using 90° light scattering to 600-nm incident radiation recorded as counts per second (cps) in HEPES buffer (10 mM HEPES, pH 7.4) at 37°C. (B) Determination of QSSM CMC using 90° light scattering to 600-nm incident radiation recorded as cps on increasing concentrations of 3-oxo-C10 HSL, (C) 3-oxo-C12 HSL(L), and (D) 3-oxo-C14 HSL. (E) The AHL derivatives C12 HSL (●), 3-OH-C12 HSL (○), and 3-oxo-C12 HS (×). (F) The AQ signaling molecules PQS (×) and HHQ (▴) in sucrose-Tris buffer (280 mM sucrose, 10 mM Tris, pH 7.4) at 37°C (n = 3, mean ± SE). Biophysical Journal 2011 101, 245-254DOI: (10.1016/j.bpj.2011.05.033) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 3 (A) An investigation into the temperature dependence of 3-oxo-C12 HSL CMC through measuring changes in 90° light scattering using 600-nm incident radiation, as temperature was decreased from 37°C to 10°C in the presence of 150-mM 3-oxo-C12 HSL (a sub-CMC concentration at 37°C, solid line) or equivalent volumes of DMSO (0.5% v/v, shaded line). (B) Determination of QSSM CMC using 90° light scattering to 600-nm incident radiation recorded as cps on increasing concentration of 3-oxo-C14 HSL in the presence of 400 μM PC 100% PLVs (○) or (C) 50 mg.mL-1 HSA (×). The light scattering for 3-oxo-C14 HSL in buffer alone is shown in each case as a solid circle (●). All experiments were conducted in sucrose-Tris buffer (280 mM sucrose, 10 mM Tris, pH 7.4) at 37°C (n = 3, mean ± SE). Biophysical Journal 2011 101, 245-254DOI: (10.1016/j.bpj.2011.05.033) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 4 Correlation function of 10 mM Tris buffer (A) or 10 mM Tris buffer in the presence of 10 μM 3-oxo-C14 HSL (B) at 37°C (pH 7.4). (C) Number distribution of particle radii obtained for 25 μM 3-oxo-C14 HSL in 10 mM Tris buffer with the corresponding correlation function (Inset). (D) Zeta potential (ξ) of 3-oxo-C14 HSL, 3-oxo-C12 HSL, and C12 HSL micelles in 10 mM Tris buffer (pH 7.4) or distilled water (dH2O) at 37°C. Where 10 mM Tris buffer was used, pH was set to the desired level using small quantities of hydrogen chloride unless otherwise specified, in which case sulphuric acid was used to change the anions present in solution to test the extent of particle anion absorption (n = 3, mean ± SE). Biophysical Journal 2011 101, 245-254DOI: (10.1016/j.bpj.2011.05.033) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 5 Rate of C10 HSL TMA production on decomposition of 200 μM 3-oxo-C12 HSL (○) or 200 μM 3-OH-C12 HSL (▴) in sucrose-Tris buffer (280 mM sucrose and 10 mM Tris, pH 7.4) at 37°C (final DMSO solvent concentration did not exceed 0.5% v/v). Rate of C10 HSL TMA production was determined through measuring AHL absorbance at 280 nm at 5 min intervals for a total of 2 h. Concentrations of C10 HSL TMA produced were determined through application of Beer-Lamberts law and the decadic molar extinction coefficient of C10 HSL TMA was measured as 13,380 M−1.cm−1 using methods described in the text (n = 3, mean ± SE). Biophysical Journal 2011 101, 245-254DOI: (10.1016/j.bpj.2011.05.033) Copyright © 2011 Biophysical Society Terms and Conditions