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 microbubbl...

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 p...

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