The antimycotic activity of amphotericin B depends on its ability to make complexes with cell membrane sterols. Surface pressure (π) as a function of surface area (A) and π-A hysteresis were measured for monolayers of amphotericin B/cholesterol mixtures on the water/air interface. Specific area per molecule of amphotericin B and free energy of mixing were calculated as a function of concentration of amphotericin B. When chloroform/methanol was used as a spreading solvent, the π-A isotherms of the mixed monolayers exhibited characteristic transitions from the gas to liquid-expanded, then liquid-condensed, and finally the solid state. The mean molecular area of the mixed monolayers was significantly higher than the calculated sum of the molecular areas of the pure components. This expanding effect was accompanied by a large π-A hysteresis and a positive excess of free energy of mixing at high π. In contrast, when 2-propanol/water was used as spreading solvent, the mixed monolayers at 20 °C exhibited π-A isotherms with no visible transitions, low hysteresis, a condensing effect, and a negative free energy of mixing. The most stable monolayers were produced from molecules of amphotericin B and cholesterol with a 2:1 stoichiometry. At this ratio, amphotericin B and cholesterol form ion channels in lipid bilayers with conductance of 4-400 pS. These results provide a better understanding of the biological activity of amphotericin B. Artificial amphotericin B/cholesterol ion channels having large conductance could be useful in nanotechnology.
Filamentous phage affinity-selected for streptavidin and Salmonella typhimurium from phage display libraries were transformed with chloroform into spherical forms then deposited as phage coat protein monolayers to quartz crystal microbalances (QCM) by Langmuir-Blodgett (LB) to prepare bioselective sensors. Maximum yield of spheroids was achieved by mixing 8.3 x 10-11 to 2.5 x 10-12 filamentous phage virions/ml with 1 ml chloroform for 60 seconds. Spheroid conversion and binding to S. typhimurium was confirmed by transmission electron microscopy. Phage coat monolayers incorporating phospholipid possessed high elasticity and transference to QCM substrates comparable to antibodies. Sensor responses to increasing concentrations of target analytes were characterized by rapid reaction, steady-state equilibrium, high sensitivity, and linear dose-response that followed mass theory for piezoelectric transducers. Scanning electron microscopy confirmed binding of target analytes to biosensors compared to controls. Phage-based sensors may allow detection of bacterial agents such as S. typhimurium in food safety and security applications.
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