Real-Time Force Measurement Between Emulsion Droplets During Enzymatic Breakdown

We used optic tweezers to measure real-time forces between individual emulsion droplets during enzyme digestion to understand the stability during digestion process. During the optic tweezer experiment, the digestion process is continuous. The optimal concentration of enzymes are difficult to determine, and if we fail to capture droplets in time, we need to restart the digestion all over again. The slope and maximum force between individual droplets are indeed tied to the oil type, digestion time, and enzyme type. It appears for digestion with protease, oil type does not affect the digestion process. Possibly an experiment that uses both spectroscopic monitoring and the force monitoring to see the link between force and changes of chemical structures.

[Narrator] To begin, prepare the sample flow cell and then open the optical tweezers instrument. Add approximately 70 microliters of deionized water to the lower objective lens and insert the sample chamber into the instrument until a click is heard. Place a drop of immersion oil on the top surface of the sample chamber. Then using the condenser knob, gently lower the condenser lens until it lightly touches the top surface of the sample chamber. Close the optical tweezers instrument to complete the setup before activating the laser. Now turn on the laser and set the power to 100%. Adjust the lower knob on the optical tweezers system in a clockwise direction to locate the laser spot, moving from bottom to top. Observe the laser spots three times and set the Z-plane between the second and third sets. Then reduce the laser power to 30%. Use the joystick to capture two droplets of similar size within the channel. Adjust the distance between the two droplets to approximately 10 micrometers. Fix the X and Y positions of the right optical trap and the Y position of the left optical trap, then mark the droplets with magenta and green boxes respectively. Now navigate to the calibration menu and select measure to begin calibration. If noise levels are excessive, repeat the measurement until satisfactory results are visible. Select apply to confirm the calibration. Next, clear the original data using the appropriate software interface and measure the force distance using the calibrated optical trap system. Finally, add the trypsin solution to the system. Adjust the settings to measure every 10 minutes, starting 10 minutes after adding the trypsin solution to dynamically monitor the emulsions using the optical tweezer system. Emulsions containing rapeseed oil and whey protein and milk fat and whey protein were analyzed using optical tweezers. For rapeseed oil emulsions without trypsin, peak interaction force increased with droplet size, reaching over 100 piconewtons at eight micrometers compared to approximately 10 piconewtons at three micrometers. A similar trend was observed for milk fat emulsion droplets. In both emulsions, the mean peak interaction force increased with droplet diameter, showing a roughly linear relationship. Droplets with a diameter of five micrometers had the highest capture count among all sizes indicating optimal trapping efficiency, while capture frequencies for three micrometer and 10 micrometer droplets were the lowest. After adding trypsin, milk fat emulsion droplets showed a gradual decline in peak interaction force from 31.9 piconewtons at 10 minutes to 6.6 piconewtons at 60 minutes. In rapeseed oil emulsions, trypsin treatment caused the force distance curves to decline sharply, suggesting that the efficiency of enzymatic action is more in the liquid oil phase.