Detection of environmental pH and oxygenation through ultrasound microbubble shell stiffening using nonlinear pulse sequences

Ultrasound microbubble (MB) contrast agents, typically consisting of a lipid shell and an inert gas core, are commonly used as tracers to highlight the vascular system. Recently, MBs with compositions suitable to sense specific biological factors, such as pH or oxygen level, have been developed. Studies have shown that MBs can undergo changes in their shell properties, such as stiffness, in response to alterations in their local environment (e.g., pH or oxygen saturation). In this work, we evaluate the response of MBs using B-mode imaging and nonlinear imaging to detect changes in pH and oxygen levels in their surrounding environment. In vitro experiments were performed with lipid-shelled MB in solutions with baseline pH values of 7.4 which were adjusted to 5.5, and with hemoglobin-shelled MB in high and low oxygen conditions. The MBs were imaged with a Verasonics Vantage 256 system, equipped with a GE9LD linear transducer at 5 MHz. The results demonstrated a 1.3× to 3× increase in harmonic content with reduced oxygenation, or lower pH, attributed to changes in the MB shell stiffness. These findings also validated simulation predictions. This study demonstrates the potential of using MBs to detect bio-environmental differences and offers a promising direction for further study.