2.4. Reaction rate on MB surface

To explore the rate of reaction between DBCO on MB surface and azide group from ligation compound, we used TAMRA-biotin-azide (1174 g mol⁻¹) with fluorescent marker as tracers. The fluorescence intensity changes as a function of incubation time were recorded and analyzed. Briefly, 0.1 mL of freshly prepared MB-DBCO microbubbles (DBCO functional group 9.6 × 10⁻⁸ mol) were diluted in 20 mL of prewarmed 5% HSA solution (37 °C) saturated with C₄F₁₀ gas. TAMRA-biotin-azide was diluted to 10 μg/μl and then 10 μl of TAMRA-biotin-azide (100 μg, 8.5 × 10⁻⁸ mol) were added. The reaction system was incubated at 37 °C under gentle shaking for various time lengths of 1 min, 3 min, 5 min, 10 min, and 30 min. Obtained microbubble product was purified using the centrifugation-flotation exchange of subnatant method, according to a previous study.⁵ Briefly, MBs after reaction were centrifuged at 500 rpm for 1 min and the subnatant (~18 mL) was discarded. Surficial bubble layer was then collected (~ 2 mL) and washed gently with the same volume (~18 mL) of cold degassed phosphate-buffered saline (PBS) once. The centrifugation-flotation process was repeated three times to fully remove unconjugated components and any bubble fragments. Purified MBs were then observed using an Axiovert 25 Zeiss light microscope (Carl Zeiss, Germany). The intensity of each bubble was analyzed using ImageJ software and normalized to background fluorescence (set as 1). For each time point, the fluorescence intensities of 150 microbubbles were recorded and averaged.