Production of Stable Monodisperse Phospholipid-coated Microbubbles at Room Temperature Using a Microfluidic Flow-focusing Device

Summary

This article describes a method to produce monodisperse phospholipid-coated microbubbles using a microfluidic flow-focusing device at room temperature, including how to prepare the phospholipid coating formulation.

Abstract

Monodisperse microbubbles have demonstrated significant potential in enhancing the efficacy of ultrasound contrast agents for both imaging and therapeutic applications. These microbubbles can be directly produced using a microfluidic flow-focusing method, which allows precise control over their size. However, preventing coalescence during production remains a critical challenge. While elevated production temperatures (e.g., 55 °C) can suppress coalescence, such conditions complicate microfluidic device design and may be incompatible with targeting agents and drug conjugates. This protocol outlines a method for producing monodisperse phospholipid-coated microbubbles at room temperature, with the microbubbles' stability and monodispersity maintained for at least 7 days. The protocol also describes the fabrication of reusable PDMS microfluidic chips and preparation of the phospholipid coating formulation containing the surfactant Pluronic F68. Our findings show that the microbubbles produced using this method maintain their size and stability over 7 days, supporting their future applicability in clinical settings where stable and controllable ultrasound contrast agents are essential.

Introduction

Ultrasound contrast agents (UCA) are widely used in clinical echography and therapeutic applications^1,2,3. Administered intravenously, these microbubbles remain confined in the vascular system, acting as blood pool agents^4,5. A stabilizing shell coats the microbubbles and reduces gas diffusion driven by Laplace pressure, thus extending the microbubble's lifespan^3,6. When exposed to ultrasound, microbubbles start to oscillate and produce strong echoes due to the compressibility of the gas core^7,8. The oscillation is most pronounced at the microbubble's resonance frequency, which is influenced by the microbubble size and shell properties^9,10,11. Clinically approved UCA generally consist of polydisperse microbubbles with a broad size distribution, typically ranging from approximately 0.5-10  µm in diameter^12,13,14. This broad size distribution leads to a wide range of frequencies at which these microbubbles are resonant. Given that clinical ultrasound probes typically operate in a narrow frequency bandwidth, only a subset of the UCA population resonates effectively¹⁵. Monodisperse microbubbles, which have a narrow size distribution (e.g., PDI 3~10%^16,17), have been shown to augment diagnosis and therapy in comparison to polydisperse microbubbles. For contrast-enhanced ultrasound imaging, the intensity increased by two to three orders of magnitude¹⁸, while the sensitivity for noninvasive pressure sensing through subharmonic signals was nearly doubled^19,20,21, and drug delivery efficiency was augmented by nearly one order of magnitude²². Consequently, producing monodisperse microbubbles with a narrow size distribution and thus acoustic response is key to maximizing their potential for ultrasound imaging and therapeutic applications, where controlled microbubble oscillations are critical.

Monodisperse microbubbles can be produced through various methods, such as decantation²³ , mechanical filtration²⁴, and centrifugation of a polydisperse microbubble agent²⁵. However, achieving precise size control and high monodispersity remains challenging with these techniques²⁶. An alternative and more controlled approach is the direct production of monodisperse microbubbles using a microfluidic flow-focusing device. In this process, a gas thread is focused between two aqueous liposome streams on a microfluidic chip, leading to reproducible pinching off of the gas thread, which allows for high-speed production of monodisperse microbubbles at desired size²⁷. Although flow-focusing methods have been widely used for producing monodisperse droplets^28,29, their application in monodisperse microbubble production is less common due to the difficulty of preventing coalescence and properly maintaining long-term stability in terms of size, monodispersity, and concentration³⁰. Coalescence, where multiple microbubbles merge into larger ones, impacts monodispersity. Elevated production temperatures have been shown to reduce coalescence³¹, but this complicates microfluidic system design and may not be suitable for formulations involving targeting agents or drug conjugates. Therefore, producing monodisperse microbubbles at room temperature would be advantageous. Equally important is the method of storing these monodisperse microbubbles, as phospholipid-coated microbubbles tend to shrink, collapse, and lose their monodispersity over time³². Ensuring long-term stability, over several days, during storage is critical for translating monodisperse microbubble technology into clinical applications.

In this protocol, we describe a method for the production of phospholipid-coated monodisperse microbubbles using a flow-focusing microfluidic platform at room temperature. This method is compatible with lipid flow rates ranging from 13-42 µL/min and is designed for use at room temperature, making it suitable for laboratories lacking temperature control on their microfluidic system. The protocol utilizes a phospholipid formulation consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carbonyl-methoxypolyethylene glycol] (DPPE-PEG5000), and Pluronic F68, which ensures stability and reproducibility. The protocol provides step-by-step instructions on fabricating reusable PDMS microfluidic chips, preparing the phospholipid coating solutions, and incorporating the surfactant Pluronic F68 to prevent microbubble coalescence during production at room temperature. It also details the procedures for cleaning the microfluidic system to prevent nozzle clogging, adjusting the gas pressure and liquid flow rate to control microbubble size, and collecting and storing the produced microbubbles for long-term stability. This method offers a practical approach for generating stable monodisperse microbubbles with precise size control at room temperature.

Protocol

1. Making lipid films

NOTE: The lipid mixture for the monodisperse microbubble production is formulated with a binary mixture of DSPC and DPPE-PEG5000 at a molar ratio of 90:10. DSPC was chosen as the main lipid because it has been shown to provide superior stability against gas dissolution in both polydisperse^33,34 and monodisperse microbubbles³¹. This enhanced stability is hypothesized to result from its relatively high gel-to-liquid crystalline phase transition temperature of 55 °C³⁵, which is higher than that of other commonly used lipids such as DPPC (~41 °C)³⁵, thereby leading to a more rigid and impermeable lipid shell at room temperature³⁶. Here, an example of preparing a 3 mL phospholipid coating solution is demonstrated. To achieve a final phospholipid concentration of 20 mg/mL, which is a typical concentration for producing monodisperse microbubbles^16,17, 60 mg of phospholipids is required. The desired amount of each phospholipid component is given in Table 1.

1. Remove the phospholipid powder from the -20 °C freezer and allow to thaw at room temperature for 1 h.
2. Weigh the needed lipid powder in separate 2 mL volumetric flasks.
3. Prepare100 mL of a 90:10 v/v mixture of chloroform:methanol solvent in a fume hoodby measuring 90 mL of chloroform using a 100 mL glass measuring cylinder and then transferring this to a 250 mL glass Erlenmeyer. Then measure 10 mL of methanol using the 100 mL glass measuring cylinder and pour this into to the 250 mL glass Erlenmeyer that already contains the chloroform. Close the glass Erlenmeyer with a glass stopper and mix the two solvents by gently swirling the Erlenmeyer until the solution has become clear.
   CAUTION: Chloroform and methanol are toxic chemicals. Chloroform is a potential carcinogen and can cause respiratory, skin, and eye irritation. Methanol is highly flammable, toxic if inhaled, ingested, or absorbed through the skin, and can cause blindness if ingested. Always work with chloroform/methanol mixtures in a well-ventilated fume hood to avoid inhalation of fumes. Wear appropriate personal protective equipment, including gloves, safety goggles, and a lab coat. Ensure that no open flames or ignition sources are present when handling methanol. Dispose of solvent waste according to the institution's hazardous waste protocols.
4. To fully dissolve the lipid powders weighed in Step 1.2, add  mL of the chloroform/methanol mixture to each 2 mL volumetric glass flask and quickly close the glass lid to prevent evaporation.
   NOTE: Ensure the lipid powder is fully dissolved. If necessary, gently swirl the flask to facilitate dissolution.
5. Mix the dissolved lipid solutions into a 30 mL glass vial by pouring each solution sequentially.
6. Evaporate the solvent under a steady flow of argon gas, rotating the vial to ensure even distribution of the lipid film on the glass surface.
   NOTE: The final traces of solvent may be difficult to evaporate using argon gas alone. Leave the vial in the fume hood for 2 h to allow further drying. If argon gas is unavailable, nitrogen gas³⁷ or a vacuum rotary evaporator operated at 6 °C^31,38 (i.e., above the phase transition temperature of DSPC of 55 °C³⁵) can be used to evaporate the chloroform/methanol solvent.
7. After solvent evaporation, subject the lipid mixture to vacuum to eliminate any remaining organic solvent residues. Seal the glass vial with a layer of parafilm and puncture several holes with a needle to allow airflow during vacuum.
   NOTE: The purpose of using parafilm is to prevent the lipid from dispersing out of the glass vial during the freeze-drying process.
8. Place the vials in a freeze-dryer under vacuum overnight to remove any residual organic solvent.
9. Proceed to the next step or store the lipid film at -20 °C until needed.
   NOTE: No centrifugation step is used in this section or elsewhere in the protocol.

2. Fabrication of flow-focusing microfluidic chips

NOTE: Monodisperse microbubbles are produced using a flow-focusing microfluidic chip, as illustrated in Figure 1A. A schematic of the microfluidic channel geometry, including nozzle and outlet dimensions, is provided in Supplementary Figure S1. A detailed fabrication protocol for SU-8-based photolithography used to prepare these molds has been described previously³⁷ and can be found as Supplementary Information to this protocol. The fabrication process of the PDMS chip is outlined in the following steps.

1. Mix the PDMS base (23. g) and curing agent (2.3 g) at a 10:1 weight ratio to obtain a total of 25.5 g of PDMS.
2. Place the master mold in a petri dish and pour the PDMS mixture over it.
3. Degas the mixture under vacuum (2~7 mbar) for 40 min to remove the trapped air bubbles.
   NOTE: For larger volumes, a longer degassing time may be required to ensure complete bubble removal.
4. Place the mold on a 75 °C hot plate for 1 h.
   NOTE: For larger volumes, a longer curing time may be required.
5. Allow the PDMS to cool to room temperature. Using a scalpel, carefully cut around the cured PDMS and peel it off to release the chip.
6. Cut the PDMS chip to the desired size and punch the inlets/outlets holes using a 1 mm punch.
7. Clean the PDMS chip with adhesive tape to remove dust particles.
8. Clean both the PDMS chip and glass slide with isopropanol, followed by deionized water.
9. Bond the PDMS to the glass slide by treating them in an air plasma cleaner for 1 min at 60% power (60 W).
10. Place the bonded chip on a 95 °C hot plate for 3 min to enhance the bonding.
11. Allow the PDMS chip to cool down to room temperature. The fully fabricated chip is shown in Figure 1C, and a microscopic bright field picture of the nozzle is shown in Figure 1D.
12. Place the bonded chip in the plasma cleaner for 5 min at 80% power (80 W) to restore the surface hydrophilicity of the microfluidic channel.
13. Immediately fill the chip with Milli-Q water to preserve hydrophilicity.
14. Store the chips at room temperature in Milli-Q water until usage.

3. Preparation of phospholipid coating solution

1. Remove the prepared lipid film from the -20 °C freezer and thaw at room temperature for 1 h.
2. Add 3 mL of pre-warmed (55 °C) phosphate-buffered saline (PBS) to the lipid film (60 mg) to reach a final lipid concentration of 20 mg/mL. Place the vials in a 55 °C water bath for 40 min, vortexing every 15 min at medium speed (level 3 of 5 on the vortex mixer).
   NOTE: This step serves to pre-dissolve and uniformly disperse the lipid components in PBS prior to sonication in step 3.5. This temperature is the phase transition temperature of DSPC³⁹, promoting partial solubilization and reducing lipid aggregation. Although sonication in step 3.6 is required to achieve full optical clarity, the preliminary thermal incubation facilitates more efficient and reproducible sonication by minimizing the formation of large lipid aggregates and enhancing the homogeneity of the dispersion.
3. Add 0.43 mL of Pluronic F68 solution to the lipid mixture to reach a molar concentration of 10%, namely, DSPC: DPPE-PEG5000: Pluronic F68 = 81: 9: 10 mol%. Afterwards, the sample should visually resemble Figure 2A, which is a cloudy solution.
   NOTE: Pluronic F68 is a surfactant used to prevent coalescence during monodisperse microbubble production^16,40. In the absence of Pluronic F68, monodisperse microbubbles cannot be formed using the Horizon microfluidic device³⁷and lipid formulation described in this protocol¹⁶. No propylene glycol was used in the lipid formulation, as this compound has been reported to compromise both the monodispersity and stability of the resulting monodisperse microbubble suspension³⁸.
4. For the preparation of fluorescently labeled monodisperse microbubbles, incorporate a lipid dye, such as a lipophilic carbocyanine dye , into the mixture at this stage.
5. Sonicate the lipid mixture in a water bath at 100% power for 20 min, maintaining the water temperature at 55 °C. The lipid solution should resemble Figure 2B after 20 min, which is a less cloudy solution than in step 3.4. Continue sonication in 10 min intervals until the solution is clear, as shown in Figure 2C.
   NOTE: For the solution in Figure 2C, 30 min of sonication was needed to achieve a clear solution. Characterization of the lipid solution can be performed by measuring the liposome size using, for example, a dynamic light scatterer.
6. Allow the lipid solution to cool to room temperature before further processing.

4. Preparation of microbubble collection vial

NOTE: To ensure long-term storage of monodisperse microbubbles, a gas tight medical vial is needed as a collection vial.

1. Seal a 5 mL glass medical vial with a rubber stopper and fill the vial with C₄F₁₀ gas. Subsequently, crimp an aluminum cap around the vial to ensure the gas tightness. The collection vial is now ready for use.

5. Preparation of the microfluidic platform

NOTE: As illustrated in Figure 3A, the system comprises the Horizon microfluidic platform³⁷ (hereafter referred to as 'microfluidic platform'), a computer for control, a high-speed camera for monitoring the production process, an external light-emitting diode (LED) light source for illumination during high-speed imaging, and a function waveform generator for triggering high-speed imaging at specific intervals during production. Before starting the microbubble production, the device must be cleaned to reduce the likelihood of nozzle clogging. The cleaning procedure involves both the tubing and the microfluidic chip.

1. Tubing cleaning procedure
   1. Turn on the power switch of the microfluidic platform, which is located on the right side (Figure 3A).
      NOTE: The "power switch" refers to the main power control unit of the Horizon platform, which activates the system's internal microcontroller, pumps, and sensors.
   2. Place a 4 mL glass vial filled with 70% ethanol into the left side of the P-pump chamber of the microfluidic platform (Figure 3). Start the liquid flow at 100 µL/min so the liquid inlet tubing will be flushed with the 70% ethanol from the vial. Stop the flushing by stopping the liquid flow after 5 min.
      NOTE: P-pump refers to the pressure-controlled gas delivery unit integrated within the microfluidic system. This step is intended to clean the tubing only; the tubing should not be connected to the microfluidic chip at this stage. This process removes ethanol-soluble dust and contaminants from the fluidic system.
   3. Replace the ethanol-filled vial with a Milli-Q water-filled vial.
   4. Flush the tubing with Milli-Q water at 100 µL/min for 5 min to remove any residual ethanol from the system.
2. Chip cleaning procedure
   1. Turn on the internal LED light switch of the microfluidic platform.
   2. Insert the PDMS chip into the holder.
   3. Position the manifold over the chip and secure it using the adjustable wheel.
   4. Attach the gas, liquid, and outlet tubing to the PDMS chip using tweezers. The connection configuration is shown in Figure 3B. Ensure that the outlet tubing leads to a vial to collect the waste solution from the cleaning process.
   5. Adjust the platform to position the nozzle of the chip within the field of view of the objective.
   6. Clean the chip with Milli-Q water for 5 min using a gas pressure of 1000 mbar and a liquid flow rate of 100 µL/min. First start the gas flow, then follow with the liquid flow.
      NOTE: This step is to clean the microfluidic chip upstream of the nozzle.
   7. Stop the liquid flow, followed by the gas flow.
   8. Reconnect the gas and outlet tubing as shown in (Figure 3C).
   9. Repeat the washing procedure with Milli-Q water for another 5 min, using a gas pressure of 1000 mbar and a liquid flow rate of 50 µL/min. First start the gas flow, then follow with the liquid flow.
      NOTE: This setup cleans the microfluidic chip downstream of the nozzle.
   10. Stop the liquid flow, followed by the gas flow. The PDMS chip is now ready for microbubble production.

6. Setup of high-speed imaging

NOTE: To monitor and control the production of monodisperse microbubbles, it is recommended to integrate a high-speed camera with the microfluidic platform, as the microbubble will be produced at a production rate higher than 10⁵ microbubbles/s. The high-speed camera allows the measurement of the on-chip microbubble size and production rate, as well as monitoring microbubble on-chip coalescence.

1. Mount an LED light source above the mounting platform (Figure 3A) to illuminate the microfluidic chip during high-speed imaging.
2. Couple the high-speed camera to the side imaging port of the microfluidic platform.
3. Utilize an external function waveform generator to trigger high-speed recordings at 10 s intervals for the entire duration of the microbubble production. Connect the TTL trigger output of the waveform generator to the external trigger input of the high-speed camera using a standard BNC cable to ensure synchronized acquisition. This monitoring is needed to assess the shape of the gas thread and the size stability of the microbubbles during production.
   NOTE: Continuous monitoring is important, as clogging may occur during production. In addition, the high-speed image sequences are used to extract time-resolved on-chip microbubble size distributions. This 10 s interval can be adjusted depending on user requirements.'

7. Production and characterization of monodisperse microbubbles

1. Prior to starting production, set up the collecting vial as shown in Figure 4A. Pierce one needle through the rubber stopper to serve as the collecting needle, and another needle through the stopper as a ventilation needle, with the tip of the needle positioned near the bottom of the vial. Keep the collecting vial inverted using a clamp during production.
   NOTE: The ventilation needle ensures constant pressure inside the vial during collection. Since C₄F₁₀ is heavier than air, it does not spontaneously exit the vial.
2. Load the prepared phospholipid solution into P-pump of the microfluidic platform.
3. Move the nozzle part of the chip to the field of view of the objective.
   NOTE: The microfluidic platform includes an internal optical module with adjustable zoom and focus, and an external F-mount coupler for attaching a high-speed camera.
4. To initiate microbubble production, set the gas pressure to 800 mbar and the liquid flow rate to 40 µL/min.
   NOTE: The microfluidic platform integrates two flow-controlled liquid channels, 1-5 µL/min on the left, and 1-100 µL/min on the right, and one pressure-controlled gas channel. In this protocol, the 1-100 µL/min liquid channel and pressure-controlled gas channel were used.
5. Once the liquid reaches the nozzle, the gas thread pinches off and releases monodisperse microbubbles through the outlet (Figure 4A).
6. Set the high-speed recording to a frame rate of ≥ 200,000 fps and use high-speed recordings to measure the size of the produced microbubbles via the imfindcircles function in MATLAB.
7. Adjust the gas pressure and liquid flow rate to control the microbubble size.
   NOTE: After adjusting the flow rate or/and gas pressure, use the MATLAB script to plot the on-chip size distribution from the high-speed recording. Fitting the size distribution with a Gaussian distribution, the mean µ and standard deviation σ can be derived. The µ represents the on-chip size of the produced monodisperse microbubbles. Microbubble coalescence might occur at the initial stage of production due to residual Milli-Q water in the tubing, which dilutes the lipid concentration. This issue typically resolves once the undiluted lipid solution reaches the nozzle. Table 2 summarizes the resulting microbubble diameters for several applied flow rates at a constant gas pressure of 800 mbar; various combinations of gas pressure and liquid flow rate can produce similar microbubble sizes, though different parameter combinations also influence the production rate. At a constant gas flow rate, it is known that the production rate is positively correlated with the liquid flow rates⁴¹. The microbubble production rate in this protocol is on the order of ~10⁵ microbubbles per second, based on a frame-by-frame analysis of the high-speed recordings, which is consistent with previously reported values for similar flow-focusing geometries³⁷.
8. Once the desired microbubble size is achieved, collect the produced microbubbles by connecting the outlet tubing to the collecting vial.
9. During collection, set the high-speed recording to trigger every 10 s to monitor the production process.
   NOTE: The interval between recordings can be adjusted based on specific requirements.
10. After sufficient microbubble suspension has been collected, remove both the ventilation needle and the collecting needle from the collecting vial.
11. Stop the liquid flow, followed by the gas flow.
12. Store the collected microbubbles upright at room temperature, ensuring the vial gets minimally mechanically agitated.
    NOTE: The microbubble solution will have three layers as shown in Figure 5A: a foam layer, a monodisperse microbubble layer, and a subnatant. It is best to leave the foam layer in the vial, as previous studies reported that foam-associated bubbles can gradually shrink to a monodisperse size^16,38. The microbubbles produced using this protocol have not undergone regulatory safety evaluation. However, the constituent lipids (DSPC, DPPE-PEG5000) and surfactant (Pluronic F68) are commonly used in FDA-approved injectable formulations and are considered biocompatible^42,43. Further preclinical evaluation is required to establish safety for medical use.
13. 2 h post-production (i.e., 2 h after production of the monodisperse microbubbles), characterize the final size and concentration of the microbubbles using a particle characterization machine.
    NOTE: Monodisperse microbubbles produced by the flow-focusing method require approximately 90 min to stabilize to their final size⁴⁴. Therefore, it is recommended to allow at least 2 h of stabilization before using the microbubbles.
14. Attach a 19 G needle to a  mL syringe, hereafter referred to as the 'extraction syringe'.
15. Insert a separate 19 G needle into the microbubble vial as a ventilation needle. Ensure the tip of the needle remains near the top of the vial to allow air entrance and maintain the internal pressure during microbubble extraction.
16. Gently swirl the vial to homogenize the suspension. The suspension will separate into three layers: a top foam layer, a middle layer containing monodisperse microbubbles, and a bottom aqueous layer.
17. Quickly insert the extraction syringe and carefully position the needle tip within the middle of the monodisperse microbubble layer, avoiding the foam and lower aqueous subnatant layer. Withdraw the desired volume of microbubble suspension and remove the extraction syringe from the vial. When microbubble extraction is complete, remove the ventilation needle.

8. Cleaning and shutdown procedure

NOTE: After microbubble production, the microfluidic platform and chip must be thoroughly cleaned to ensure proper functioning and allow the reuse of the chip. The following steps outline the cleaning process.

1. For the chip, proceed as follows:
   1. Replace the lipid vial with a Milli-Q water vial.
   2. Flush the microfluidic chip using a gas pressure of 800 mbar and a liquid flow rate of 40 µL/min for 5 min.
   3. Stop the liquid flow first, followed by stopping the gas flow.
   4. Disconnect only the gas inlet tubing from the microfluidic chip.
   5. Fill the microfluidic chip with Milli-Q water to maintain the hydrophilicity of the internal surfaces.
   6. Stop the liquid flow and disconnect all remaining tubing from the chip.
   7. Store the chip in Milli-Q water until it is needed for future use.
2. For the microfluidic system, proceed as follows:
   1. Replace the Milli-Q water vial with a 70% ethanol vial in the microfluidic platform.
   2. Flush the liquid inlet tubing with 70% ethanol at a flow rate of 100 µL/min for 5 min to remove any lipid residues.
   3. Replace the ethanol vial with a Milli-Q water vial in P-Pump1.
   4. Flush the tubing with Milli-Q water at 100 µL/min for 5 min to remove any residual ethanol from the system. The system is now clean.
   5. Shut down the microfluidic platform and high-speed camera system.

9. Troubleshooting steps

1. Nozzle blockage
   NOTE: Given the small size of the nozzle (5 µm), blockages can occur during the production process. To clear the blockage, follow the steps below.
   1. Immediately stop the liquid flow, followed by the gas flow.
   2. Reconnect the tubing according to the setup shown in Figure 3B.
   3. Replace the phospholipid-filled vial with a Milli-Q water-filled vial in P-Pump1.
   4. Apply a gas pressure of 1000 mbar and set the liquid flow rate to 100 µL/min for 5 min to flush the nozzle.
      NOTE: In most cases, blockages are cleared after a 5 min rinse. However, if the blockage persists, it may be necessary to replace the PDMS chip with a new one.
   5. Once the blockage is cleared, reconnect the tubing as indicated in Figure 3C.
   6. Flush the chip again with Milli-Q water for 5 min, using a gas pressure of 1000 mbar and a liquid flow rate of 50 µL/min to ensure complete clearance.
   7. Replace the Milli-Q water-filled vial with the phospholipid-filled vial in P-Pump1, and resume microbubble production.
      NOTE: If blockages persist during production, this may indicate insufficient cleaning of the tubing or microfluidic chip. In this case, disconnect the tubing from the chip and follow the cleaning procedure outlined in step 5. If blockages continue to occur after thoroughly cleaning the entire system, it could suggest a failure of the internal filter in the microfluidic platform. Replacing the filter with a new one may resolve the issue.
2. Reuse the microfluidic chip if desired.
   NOTE: If the chip is not stored properly, it may lose hydrophilicity. If this occurs, the pinch-off of the gas thread may resemble the pattern shown in Figure 5E. In such cases, it is recommended to switch to a new chip that is hydrophilic to ensure consistent and reliable microbubble production.

Results

In this protocol, we present a method for producing stable, monodisperse microbubbles at room temperature using a flow-focusing microfluidic chip. The PDMS chip is fabricated by casting from a master mold, as shown in Figure 1. To bond the PDMS chip to a glass slide, plasma treatment is employed. Plasma treatment is crucial as it alters the PDMS surface from hydrophobic to hydrophilic. This hydrophilicity is essential for maintaining the shape and position of the gas thread during microbubble production, enabling long-term stable generation (on the order of hours) of monodisperse microbubbles at a consistent size (Figure 5D). Specifically, in a hydrophilic chip, the gas thread remains geometrically stabilized because it anchors into the corners of the gas inlet channel through capillary forces, thereby preserving a stable and symmetric pinch-off gas thread geometry. If the PDMS surface loses its hydrophilicity, the gas thread (highlighted by white arrows in Figure 5E) no longer anchors into the gas inlet channel corners (indicated by orange arrows in Figure 5E). As a consequence, the gas thread becomes prone to positional drifts⁴⁵, which is known to alter the gas thread geometry and disrupt the pinch off dynamics^45,46. These gas thread geometry alterations lead to size fluctuations in the formed monodisperse microbubbles-even when the applied gas pressure and liquid flow rate remain constant. Consequently, while microbubbles still appear monodisperse during production, their diameters can vary throughout the production period, thereby compromising the overall monodispersity of the batch.

To prepare the lipid solution for monodisperse microbubble production, the rehydrated lipid film must undergo sonication to reduce the size of the liposomes. Proper sonication results in a clear lipid solution, as illustrated in Figure 2. An unclear lipid solution indicates the presence of large liposomes. The used microfluidic platform has an internal filter that is integrated within the liquid flow path as internal non-accessible component of the Horizon platform³⁷, which has a pore size of 0.5 µm to remove impurities and reduce the risk of nozzle blockage. However, large liposomes are likely to be filtered out, leading to lipid wastage and blockages. Therefore, it is crucial to sonicate the lipid solution until it reaches the desired clarity, as shown in the figure, to ensure optimal use of the lipid and efficient microbubble production.

The typical process for producing monodisperse microbubbles is illustrated in Figure 4. In this process, a C₄F₁₀ gas thread is focused by two perpendicular lipid liquid flows as it passes through the nozzle, continuously pinching off to release monodisperse microbubbles. The size of the microbubbles formed on-chip is controlled by the gas pressure, liquid flow rate, and the geometry of the chip's nozzle. In the used microfluidic chip, the chip's nozzle has a width of 5 µm and a depth of 24.5 µm. In our experiment, a constant gas pressure of 800 mbar was maintained, and the liquid flow rate was adjusted to control the size of the microbubbles produced on-chip. Figure 4B-E demonstrates the varying on-chip radius of microbubbles produced at different flow rates.

Freshly formed lipid coated monodisperse microbubbles are inherently unstable and undergo post-production shrinkage due to the Laplace pressure^16,44,47. Figure 4B-E shows the measured size distribution of the produced monodisperse microbubbles after 1 d, illustrating their shrinkage. In our experiments, using a lipid mixture of DSPC: DPPE-PEG5000: Pluronic F68 at a molar ratio of 81:9:10, the shrinkage ratio was around 3.0, defined by R_on-chip/R_final, where R_on-chip is the radius of the microbubble on the chip and R_final is the radius of the microbubble at 1 d. Studies have shown that the shrinkage ratio is solely dependent on the lipid formulation^16,31,38.

During microbubble stabilization, gas transfers between microbubbles due to Ostwald ripening, which leads to the formation of a foam layer. As a result, the collected monodisperse microbubble suspension will separate into three distinct layers after gentle mixing and set to rest, as shown in Figure 5A. The top layer consists of foam bubbles, the middle opaque layer contains the stabilized monodisperse microbubbles, and the bottom layer is the subnatant.

The produced microbubbles were stored in a gas-tight medical vial prefilled with C₄F₁₀ gas in the headspace. Since the microbubbles have a C₄F₁₀ gas core, storing them in a gas-tight environment with C₄F₁₀ helps minimize microbubble collapse and extends their shelf stability. Figure 5B shows the size distributions of monodisperse microbubbles ranging from production to 7 d of storage. During storage, the microbubble remained stable with their size decreasing by less than 2%. The monodispersity of the microbubbles gradually decreased, with the coefficient of variation (CoV) increasing around 3% (Figure 5C). There was an increase in microbubble concentration from 2 h to its peak at 1 d post-production, followed by a decrease to 36% of the maximum concentration at 7 d post-production. This initial, temporary rise in microbubble concentration is consistent with previous publications and is attributed to the shrinkage of foam bubbles into stable monodisperse microbubbles^44,48.

The produced monodisperse microbubbles can for example be used to study subharmonic responses, offering significant potential for applications like non-invasive blood pressure measurements. Figure 6A shows an example of scattering measurements comparing monodisperse and polydisperse microbubbles, conducted at a peak negative pressure (PNP) of 350 kPa at a 3.5 MHz ultrasound frequency¹⁶. The scattered frequency spectra include two monodisperse microbubble types (F1-10PF, blue line; F2-10PF, red line) and a clinically available polydisperse microbubble (green line). At this pressure, all three exhibited subharmonic responses at 1.75 MHz. Notably, while the monodisperse microbubbles showed a 2 dB lower fundamental amplitude at 3.5 MHz compared to SonoVue, their subharmonic scattering amplitude at 1.75 MHz was 6 dB higher. This 10 dB higher subharmonic-to-fundamental ratio for the monodisperse microbubbles improves the signal-to-noise ratio in comparison to the polydisperse microbubbles. As a consequence, monodisperse microbubbles offer a distinct advantage for more precise and reliable non-invasive pressure measurements.

Monodisperse microbubbles are also valuable for studying ultrasound-mediated drug delivery. Since microbubbles exhibit their highest oscillation amplitude at their resonance frequency, matching the microbubble radius to the applied ultrasound frequency can potentially enhance drug delivery efficiency. As demonstrated by an example in Figure 6B, endothelial cells were cultured in a CLINIcell to form a confluent monolayer. Monodisperse microbubbles stained with DiD, were produced with a final radius of 2.07 µm, which closely aligns with the resonance radius of 2.2 µm for DSPC-based microbubbles at 2 MHz ultrasound⁹. As shown in Figure 6B I, prior to ultrasound exposure, the microbubbles were introduced into the CLINIcell and floated against the endothelial cells. The cells were stained with Hoechst to visualize nuclei, CellMask Green to label the cell membrane, and propidium iodide (PI) to detect sonoporation. Upon ultrasound application (2 MHz, 220 kPa, 10 cycles), a 16 µm² perforated membrane area appeared at the cell edge, rapidly expanding to 102 µm² within 32 s (Figure 6B I-V). Concurrently, the PI signal reached a plateau in 32 s. From 32 s to 200 s, the perforated area increased more gradually to 162 µm², while the PI signal remained stable (Figure 6B VI).

Figure 1: Casting and bonding of the PDMS microfluidic flow focusing chip. (A) Schematic diagram illustrating the assembly process of the microfluidic chip. (B) The master mold used for fabricating the microfluidic chip. The scale bar represents 3 mm. (C) The fabricated PDMS chip, including punched holes. (D) Brightfield microscopic picture of the nozzle structure in the flow-focusing chip. The scale bar represents 20 µm. Please click here to view a larger version of this figure.

Figure 2: Example of the prepared lipid solution. (A) Hydrated lipid film before sonication, containing a lipid dye (blue color). (B) Lipid solution after 20 min of sonication in a water bath at 55 °C. (C) Lipid solution after 30 min of water bath sonication at 55 °C. The solution should become translucent after sufficient sonication. Please click here to view a larger version of this figure.

Figure 3: Configuration of the microfluidic system. (A) The setup of the experimental system for the production of monodisperse microbubbles. The microfluidic chip is situated in the microfluidic chip stage. The light blue circle indicates the power switch of the microfluidic platform. The two purple circles indicate the P-pump chamber of the microfluidic platform. (B) Tubing connection configuration for cleaning the microfluidic chip. (C) Tubing connection configuration for the production regime of monodisperse microbubbles. Please click here to view a larger version of this figure.

Figure 4: Production of monodisperse microbubbles. (A) Schematic illustration of the flow-focusing microfluidic chip used for monodisperse microbubble production (not to scale). The number weighted size distribution of monodisperse microbubbles, produced at a constant gas pressure of 800 mbar, is shown both on-chip (dark blue histogram) and 24 h post-production (orange line) for lipid liquid flow rates of: (B) 42 µL/min (1.5 µm radius), (C) 36 µL/min (2.2 µm radius), (D) 28 µL/min (2.8 µm radius), and (E) 13 µL/min (2.9 µm radius). (A-E) This image has been modified with permission from Wang et al. 2025 (https://pubs.acs.org/doi/10.1021/acsami.4c18844)¹⁶. Please click here to view a larger version of this figure.

Figure 5: Storage of produced monodisperse microbubbles. (A) Monodisperse microbubble suspension immediately after production, gently mixed, and set to rest for 5 min. (B) Number weighted size distribution of monodisperse microbubbles on-chip (dark blue histogram) and up to 7 days post-production (colored lines). (C) Change in the coefficient of variation (CoV) of produced monodisperse microbubbles over 7 days, with the shaded region indicating the standard deviation. (D) Illustration of the gas thread shape when the PDMS microfluidic surface is hydrophilic. (E) Illustration of the gas thread shape when the PDMS microfluidic surface is hydrophobic. The white arrows indicate the corners of the triangular gas thread, and the orange arrows indicate the corner of the microchannel. (B-C) images have been modified with permission from Wang et al. 2025 (https://pubs.acs.org/doi/10.1021/acsami.4c18844)¹⁶. The reader is referred to the Wang et al. 2025 publication¹⁶ for illustrations that Pluronic F68 is needed to make coalescence-free and stable monodisperse microbubbles using the Horizon microfluidic device³⁷ and lipid formulation described in this protocol. (D-E) The scale bar represents 50 µm. Please click here to view a larger version of this figure.

Figure 6. Acoustic Response and Drug Delivery Efficacy of Monodisperse Microbubbles. (A) Subharmonic scattering response of DSPC-coated monodisperse microbubbles compared to a clinically available microbubble, measured using a 30-cycle, 350 kPa ultrasound pulse at 3.5 MHz. Each plot was averaged over 32 repeated acquisitions and log compressed to minimize signal variability and noise. This image has been reproduced with permission from Wang et al. 2025 (https://pubs.acs.org/doi/10.1021/acsami.4c18844)¹⁶. (B) Drug delivery to in vitro cultured endothelial cells using a 2.2 µm monodisperse microbubble: I: Confocal microscopy image showing the initial state of endothelial cells before ultrasound treatment, with nuclei stained by Hoechst (blue), cell membranes labeled with CellMask Green (green), propidium iodide (PI) as the model drug (red), and monodisperse microbubbles stained with a lipid dye (white); II: Microbubble radius over time, derived from ultra-high-speed imaging during ultrasound exposure (220 kPa, 10 cycles, 2 MHz); III-V: Three selected frames of confocal microscopy time-lapse imaging after ultrasound, demonstrating cell membrane perforation and PI uptake; VI: The perforated cell membrane area (black with thick line representing the moving average) and intracellular PI fluorescent intensity (red with thick line representing the previously reported fit^49,50) during the timelapse recording after ultrasound. Scale bar represents 10 µm. Please click here to view a larger version of this figure.

Ingredients	Molecular weight (g/mol)	Mol%	Mol per batch	Weight (mg)
DSPC	790.2	90%	4.20×10-05	33.18
DPPE-PEG5000	5749.9	10%	4.67×10-06	26.82
Total		100%	4.67×10-05	60

Table 1: Phospholipid composition for monodisperse microbubble production. The table lists the molecular weights, molar ratios, and corresponding weights of DSPC and DPPE-PEG5000 used to prepare a 60 mg phospholipid mixture for stable monodisperse microbubble generation.

Gas pressure (mbar)	Liquid flow rate (µL/min)	On-chip radius (µm)	Radius 24-h post-production (µm)
800	42	5.6	1.5
800	36	6.2	2.2
800	28	7.9	2.8
800	13	9.6	2.9

Table 2. Overview of gas pressure and liquid flow rates for the DSPC, DPPE-PEG(5000), and Pluronic F68 coating formulation in relation to the produced microbubble sizes.

Supplementary Information S1: Fabrication of the master mold. Step-by-step description of the photolithography process used to fabricate SU-8-based masters for PDMS chip replication. Parameters for spin-coating, soft bake, exposure, post-exposure bake, and development are provided to enable reproducibility. Please click here to download this File.

Supplementary Figure S1: Schematic of the chip design. Schematic of the microfluidic chip design. The table summarizes the key channel dimensions, including orifice width, outlet length, and channel depth. Please click here to download this File.

Discussion

This protocol describes in detail how to produce and store stable monodisperse microbubbles at room temperature using a microfluidic flow-focusing chip. The monodisperse microbubble can be used for contrast-enhanced ultrasound imaging¹⁸, non-invasive pressure sensing¹⁹, and microbubble-mediated drug delivery²², both in vitro and in vivo.

The stabilization of monodisperse microbubbles post-production has previously been reported to require at least 90 min⁴⁴. This stabilization period results in the formation of a substantial foam layer, primarily due to Ostwald ripening^44,47,48. The foam layer consists of large bubbles, with some exceeding 100 µm in diameter. Although incorporating highly soluble gases, such as CO₂, into the gas core could potentially mitigate the formation of this foam layer⁴⁸, this approach is incompatible with the used microfluid device³⁷. The current microfluidic platform is equipped with only a single gas control pump and does not support dual gas inputs or independent flow rate regulation. Precise control of CO₂ concentration within the microbubbles would require two gas pumps operating under flow-based control, whereas the current system permits only pressure-based gas regulation. Over time, these larger foam bubbles gradually shrink to the final stable size of monodisperse microbubbles. This shrinkage, driven by gas diffusion out of the microbubbles, occurs because the amount of coating lipid remains unchanged throughout the Ostwald ripening process⁴⁴. Therefore, this shrinkage of foam bubbles accounts for the observed increase in the concentration of monodisperse microbubbles over the course of a 7-day storage period shown in Figure 5B.

Effective cleaning is a critical step in maintaining the stable production of monodisperse microbubbles, as it minimizes the risk of clogging. In this protocol, the microfluidic platform was cleaned with ethanol followed by Milli-Q water, while the PDMS chip was cleaned exclusively with Milli-Q water. This distinction is crucial because ethanol causes swelling of PDMS⁵¹, leading to deformation of the chip's structure. Avoiding ethanol exposure ensures that the PDMS chip maintains its structure, which is necessary for repeated use in microfluidic applications.

PDMS is widely used in the fabrication of microfluidic platforms due to its flexibility and ease of use in rapid prototyping⁵². However, this flexibility becomes a drawback under high-pressure conditions, as it can lead to the deformation of microchannels. In particular, nozzle deformation can significantly influence the size of the produced monodisperse microbubbles. In this protocol, we applied a gas pressure of 800 mbar and did not observe significant deformation. However, higher pressures may be necessary to further increase production rates, which would reduce the collection time during microbubble generation. While PDMS is suitable for prototyping, the use of more rigid materials, such as glass⁵³ or poly(methyl methacrylate) (PMMA)³⁷, is recommended for applications requiring a higher production rate. Glass is particularly advantageous, as its natural hydrophilicity eliminates the need for additional surface treatment, thus simplifying the fabrication process⁵².

To preserve the hydrophilicity and ensure the reusability of the PDMS microfluidic chip, we recommend storing the chip in Milli-Q water after each experiment. This method effectively maintains the chip's surface properties, allowing consistent performance in subsequent experiments.

Efforts to enhance the throughput of monodisperse microbubble production have led to the exploration of multichannel microfluidic designs^54,55. However, these designs present challenges, as interference between production channels can disrupt uniformity in bubble size^41,56. Achieving high throughput with parallel structures while maintaining monodisperse microbubble size remains a significant challenge that warrants further investigation.

In our protocol, Pluronic F68 was added to prevent microbubble coalescence during production at room temperature, and no coalescence was observed throughout the process. By contrast, using the same protocol without the addition of Pluronic F68 leads to rapid microbubble coalescence and unstable microbubbles in storage¹⁶. Being a synthetic triblock copolymer composed of two hydrophilic polyethylene oxide (PEO) chains and one hydrophobic polypropylene oxide (PPO) block, Pluronic F68 incorporates into phospholipid monolayers^57,58. As a result, Pluronic F68 prevents coalescence because it acts as a steric stabilizer of the microbubble shell. Producing microbubbles at room temperature may alter their properties compared to working above the lipid phase transition temperature, as temperature is known to affect the microstructure of the microbubble shell ^59,60, and such microstructural changes are related to differences in the acoustic response as previously reported by our group⁹. Increasing the production temperature above the phase transition temperature is another approach known to prevent microbubble coalescence³¹. However, this method poses challenges, particularly when the microbubbles are intended for use with targeting agents or drug payloads that are sensitive to elevated temperatures. One potential solution could involve post-processing the produced monodisperse microbubbles to functionalize them with targeting agents or drugs⁶¹. However, maintaining the monodispersity of the microbubbles during handling and functionalization remains a significant challenge. In this context, the inclusion of Pluronic F68 in the production process provided an effective means of producing biotinylated monodisperse microbubbles at room temperature, thus offering the possibility of generating avidin-targeted monodisperse microbubbles in a single step.

We believe that this article will be helpful for researchers seeking to produce stable, monodisperse phospholipid-coated microbubbles at room temperature using a microfluidic flow-focusing device, particularly for applications in contrast-enhanced ultrasound imaging, microbubble-mediated drug delivery, and non-invasive pressure sensing.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 mm biopsy punch	Kai Medical	143831
1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine (DiD)	Thermo Fisher Scientic	D307	a lipophilic carbocyanine dye
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carbonyl-methoxypolyethylene glycol] (DPPE-PEG5000)	Lipoid	CAS-No. 384835-61-4
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)	Lipoid	CAS-No. 816-94-4
100 mL glass measuring cylinder	BLAUBRAND
2 mL volumetric glass flasks	BLAUBRAND
250 mL glass Erlenmeyer	BLAUBRAND
30 mL glass vial	DWK Life Sciences	SCERC40752700D0C2
70% ethanol
75 °C hot plate
Adhesive tape
Aluminum cap	Sigma-aldrich	27016
Argon gas	Linde Gas Benelux
C4F10 gas	F2 Chemicals
Chloroform	Merck KGaA	67-66-3
Clinicallly available  ultrasound contrast agent (i.e., microbubble)	Bracco, Plan-LesOuates, Switzerland	SonoVue	Figure 6A.
Collection and ventilation 19G needle	100 Sterican	4657799
Coulter Counter Multisizer 3	Beckman Coulter	Particle characterization machine
Custom-made ultrasound contrast agent (i.e., monodisperse microbubble)	NA	F1-10PF	Figure 6A. Produced as described by: Wang, Y. et al. Influence of Pluronic F68 on Size Stability and Acoustic Behavior of Monodisperse Phospholipid-Coated Microbubbles Produced at Room Temperature. ACS Appl. Mater. Interfaces 2025, 17, 8976−8986. (2025).
Custom-made ultrasound contrast agent (i.e., monodisperse microbubble)	NA	F2-10 PF	Figure 6A. Produced as described by: Wang, Y. et al. Influence of Pluronic F68 on Size Stability and Acoustic Behavior of Monodisperse Phospholipid-Coated Microbubbles Produced at Room Temperature. ACS Appl. Mater. Interfaces 2025, 17, 8976−8986. (2025).
Demi water
External LED light	GSVITEC	GS02370
Freeze dryer	Mertin Christ GmbH	Alpha 1–2 LD plus
Function waveform generator	Agilent	33220A
Gas tight glass vial	Sigma-aldrich	Z113964-288EA
Glass slide	VWR	631-1552
High-speed camera	Photron	Nova S16
Horizon microfluidic platform	University of Leeds, England		The platform is commercially available and described in detail in: Abou-Saleh, R. H. et al. Horizon: Microfluidic platform for the production of therapeutic microbubbles and nanobubbles. Review of Scientific Instruments. 92 (7), 074105 (2021).
Matlab	The MathWorks	R2023a
Methanol	Merck KGaA	67-56-1
Milli-Q water
PDMS gel	SYLGARD	SYLGARD® 184
Phosphate-buffered saline (PBS 1x)	Gibco	10010023
Plasma cleaner	Henniker Scientific	HPT-100
Pluronic F68	Thermo Fisher Scientific	24040032
Rubber stopper	Sigma	Z166065-100EA
Tubing	Masterflex	MFLX06407-41	PTFE, 1/32" ID x 1/16 " OD; 25 ft
Tweezer
Vortex	Sigma-aldrich	Z654779
Water bath sonicator	EMAG	EMMI 30HC