Dependence of ultrasonic parameters on bovine serum albumin-toluidine blue O binding: A spectroscopic analysis

Ultrasound is increasingly explored to regulate protein-small molecule interactions, yet the relative importance of acoustic parameters under practical liquid sonication remains unclear. This study used a bovine serum albumin-toluidine blue O (BSA-TBO) model system to quantify parameter-dependent spectroscopic responses after establishing a baseline binding state. A 20 kHz probe sonicator recorded changes in the fluorescence peak intensity of bovine serum albumin and BSA-TBO as a function of treatment time, ultrasonic power, and duty cycle, while a bath sonicator qualitatively compared peak-intensity responses at 25, 40, and 59 kHz. Temperature-matched heating controls, together with circular dichroism and synchronous fluorescence measurements, were used to test whether the parameter-dependent fluorescence response was dominated by bulk thermal effects. Under the present setup and geometry, ultrasonic power was the dominant factor; duty cycle and treatment time acted as secondary modulators via temporal delivery and cumulative exposure, whereas frequency effects were weaker and non-monotonic across the tested discrete points. These results provide guidance for parameter selection and optimization in ultrasound-regulated protein-ligand systems.