Microbubble Array Research Articles

Acoustically Actuated Flow in Microrobots Powered by Axisymmetric Resonant Bubbles

Bubble‐driven microsystems inspired by the therapeutic use of microbubbles under clinical ultrasound actuation offer innovative remote manipulation in biological settings. Ultrasound‐powered microrobots, benefiting from a distribution of vibrating microbubbles (1–100 μm in diameters), exhibit localized fluid flow for self‐propulsion. Microbubbles also contribute to the design of acoustic metamaterials, where distributed actuation of bubbles enables exotic material properties (e.g., negative refractive index). Herein, a metamaterial‐inspired microrobot design, which exploits the streaming induced by the collective oscillation of microbubble arrays (> 10), is reported. Such a large distribution of bubbles offers twofold advantages: a mesoscale microrobot allowing ease of handling and motion at a considerably low acoustic driving pressure of 50 kPa. In this work, the oscillatory amplitude of a single bubble that acts as a unit cell of the metamaterial‐based microrobot is first characterized. Thereon, this amplitude is related to the flow generated by the collective vibration of all the bubbles close to the theoretically predicted resonant frequency of the bubbles. Finally, the hovering motion of the microrobot induced by the streaming and flow‐assisted debris clearance is demonstrated. Such auxiliary functionalities of the microrobot can be useful for applications like contactless sample extraction in inaccessible biological environments.

Flow-drag reduction performance of a resident electrolytic microbubble array and its mechanisms

This work examines the problem of stable high-efficiency flow drag reduction for underwater vehicles using microbubble arrays. To this end, test pieces for micropore arrays capable of forming self-stabilizing arrays of microbubbles via electrolysis have been prepared. The quantitative relationships between the electrolysis voltage, micropore size, and flow velocity (v) and the growth behaviors and residence stability of electrolyzed microbubbles were examined by experimental and numerical methods. The flow drag reduction performance and mechanisms of microbubble arrays were investigated. It has been shown that an electrode-wall micropore array can be used to achieve self-adaptive start-stop control of microbubble electrolysis. A micropore diameter-to-depth ratio of k = 2 was found to be optimal for generating arrays of electrolytic microbubbles with good stability in flow. Two types of microbubbles are generated: the air-film type exhibits greater resident stability than the protruding type. The average measured flow-drag-reduction rate of a microbubble array with 250 μm micropores was found to be around 26%. The energy-loss region of free-fluid flow with a solid wall was 0.037 mm thicker than that with a microbubble-array wall (v = 0.5 m/s), meaning that more energy is consumed, leading to greater flow drag. Significant up-thrown flow induced by elastic deformation of microbubbles and air/water interface forces can suppress turbulent coherent structural bursts and reduce near-wall Reynolds shear stress. The mean values of turbulence-related characteristic parameters (vorticity = 890.55 s−1, kinetic energy = 0.08096 m2/s2, shear stress = 17.75 Pa, and v = 0.5 m/s) of the boundary-layer flow over the microbubble-array wall were found to be greatly reduced. This demonstrates the good turbulent friction drag reduction performance of the resident microbubble array.

Amplification of Acoustic Forces Using Microbubble Arrays Enables Manipulation of Centimeter-Scale Objects.

Manipulation of macroscale objects by sound is fundamentally limited by the wavelength and object size. Resonant subwavelength scatterers such as bubbles can decouple these requirements, but typically the forces are weak. Here we show that patterning bubbles into arrays leads to geometric amplification of the scattering forces, enabling the precise assembly and manipulation of cm-scale objects. We rotate a 1cm object continuously or position it with 15 μm accuracy, using sound with a 50cm wavelength. The results are described well by a theoretical model. Our results lay the foundation for using secondary Bjerknes forces in the controlled organization and manipulation of macroscale structures.

Development of a functional salivary gland tissue chip with potential for high-content drug screening

Radiation therapy for head and neck cancers causes salivary gland dysfunction leading to permanent xerostomia. Limited progress in the discovery of new therapeutic strategies is attributed to the lack of in vitro models that mimic salivary gland function and allow high-throughput drug screening. We address this limitation by combining engineered extracellular matrices with microbubble (MB) array technology to develop functional tissue mimetics for mouse and human salivary glands. We demonstrate that mouse and human salivary tissues encapsulated within matrix metalloproteinase-degradable poly(ethylene glycol) hydrogels formed in MB arrays are viable, express key salivary gland markers, and exhibit polarized localization of functional proteins. The salivary gland mimetics (SGm) respond to calcium signaling agonists and secrete salivary proteins. SGm were then used to evaluate radiosensitivity and mitigation of radiation damage using a radioprotective compound. Altogether, SGm exhibit phenotypic and functional parameters of salivary glands, and provide an enabling technology for high-content/throughput drug testing.

Spatial ultrasound modulation by digitally controlling microbubble arrays

Acoustic waves, capable of transmitting through optically opaque objects, have been widely used in biomedical imaging, industrial sensing and particle manipulation. High-fidelity wave front shaping is essential to further improve performance in these applications. An acoustic analog to the successful spatial light modulator (SLM) in optics would be highly desirable. To date there have been no techniques shown that provide effective and dynamic modulation of a sound wave and which also support scale-up to a high number of individually addressable pixels. In the present study, we introduce a dynamic spatial ultrasound modulator (SUM), which dynamically reshapes incident plane waves into complex acoustic images. Its transmission function is set with a digitally generated pattern of microbubbles controlled by a complementary metal–oxide–semiconductor (CMOS) chip, which results in a binary amplitude acoustic hologram. We employ this device to project sequentially changing acoustic images and demonstrate the first dynamic parallel assembly of microparticles using a SUM.

Cell Lysis Based on an Oscillating Microbubble Array.

Cell lysis is a process of breaking cell membranes to release intracellular substances such as DNA, RNA, protein, or organelles from a cell. The detection of DNA, RNA, or protein from the lysed cells is of importance for cancer diagnostics and drug screening. In this study, we develop a microbubble array that enables the realization of multiple cell lysis induced by the shear stress resulting from the individual oscillating microbubbles. The oscillating microbubbles in the channel have similar vibration amplitudes, and the intracellular substances can be released from the individual cells efficiently. Moreover, the efficiency of cell lysis increases with increments of input voltage and sonication time. By means of DNA agarose-gel electrophoresis, a sufficient extraction amount of DNA released from the lysed cells can be detected, and there is no significant difference in lysis efficiency when compared to cell lysis achieved using commercial kits. With the advantages of the simple manufacturing process, low cost, high efficiency, and high speed, this device can serve as an efficient and versatile tool for the single-cell sequencing of cell biology research, disease diagnosis, and stem cell therapy.

Biomedical Ultrasound Bioeffects: Sonoporation of Cells by a Parallel Stable Cavitation Microbubble Array (Adv. Sci. 17/2019)

Biomedical Ultrasound Bioeffects: Sonoporation of Cells by a Parallel Stable Cavitation Microbubble Array (Adv. Sci. 17/2019)

Sonoporation of Cells by a Parallel Stable Cavitation Microbubble Array.

Sonoporation is a targeted drug delivery technique that employs cavitation microbubbles to generate transient pores in the cell membrane, allowing foreign substances to enter cells by passing through the pores. Due to the broad size distribution of microbubbles, cavitation events appear to be a random process, making it difficult to achieve controllable and efficient sonoporation. In this work a technique is reported using a microfluidic device that enables in parallel modulation of membrane permeability by an oscillating microbubble array. Multirectangular channels of uniform size are created at the sidewall to generate an array of monodispersed microbubbles, which oscillate with almost the same amplitude and resonant frequency, ensuring homogeneous sonoporation with high efficacy. Stable harmonic and high harmonic signals emitted by individual oscillating microbubbles are detected by a laser Doppler vibrometer, which indicates stable cavitation occurred. Under the influence of the acoustic radiation forces induced by the oscillating microbubble, single cells can be trapped at an oscillating microbubble surface. The sonoporation of single cells is directly influenced by the individual oscillating microbubble. The parallel sonoporation of multiple cells is achieved with an efficiency of 96.6 ± 1.74% at an acoustic pressure as low as 41.7 kPa.

Microbubbles: A high-throughput platform for the sustained culture, characterization, and isolation of single cells based on morphology, cytokine production, and antibody secretion.

Abstract Analysis of lymphocytes in bulk masks the heterogeneity of cellular processes. Through advancements in single cell technologies finer resolution and the true complexities of cell populations can be revealed. A major limitation of single cell technologies, especially those that enable high-throughput screening is the poor survival and proliferation of individual cells when binned separately in vitro. We have developed a novel platform technology, termed microbubbles which are scalable arrays of individual nanoliter size wells of unique spherical architecture and gas exchange properties that enable single cells to rapidly condition their microenvironment and enable long-term culture. Additionally, morphological heterogeneity is maintained in the microbubbles, unlike in conventional tissue culture wells. Microbubble arrays are compatible with conventional fluorescent microscopes and morphology and cell surface protein expression can be easily monitored longitudinally. Secreted cellular products such as antibodies and cytokines accumulate and are readily detected from cells in real-time using fluorescent probes. Arrays with 100,000 microbubbles, contained within a footprint of a standard microscope slide, can be screened using custom software in brightfield and three fluorescent channels within 10 minutes, and rare cells of interest identified based on morphological and/or functional properties are easily recovered for downstream analysis. Applications of microbubbles developed thus far include identification and isolation of rare antibody secreting cells based on antibody-specificity for monoclonal antibody development, monitoring T cell cytokine production, and characterizing cancer stem cells.

Identifying drug resistant cancer cells using microbubble well arrays.

Drug resistance is a characteristic of tumor initiating cells that can give rise to metastatic disease. In this work we demonstrate the use of microbubble well arrays as a cell culture platform to enumerate and characterize drug resistant cells in a human derived tumorigenic squamous cell carcinoma cell line. The spherical architecture and compliant hydrophobic composition of the microbubble well favors single cell survival, clonal proliferation and formation of spheres that do not grow on standard tissue culture plastic and are resistant to cisplatin. Spheres form in isolation and in microbubble wells containing proliferating cells and to some degree they stain positive for common stem cell markers CD44 and CD133. Spheres are also observed in cellularized primary human tumors cultured in microbubble arrays. This proof-of-concept study illustrates the potential for microbubble array technology to enumerate cancer cells resistant to standard care drugs with the ability to test alternative drug combinations. This capability can be developed for designing patient specific treatment strategies. Recovery of drug-resistant cells will allow amore full characterization of their gene expression profile thereby expanding our fundamental knowledge and abilityto develop new targets to fight metastatic disease.

Microbubble array diffusion assay for the detection of cell secreted factors.

The therapeutic potential of monoclonal antibodies (mAbs) makes them an ideal tool in both clinical and research applications due to their ability to recognize and bind specific epitopes with high affinity and selectivity. While mAbs offer significant therapeutic potential, their utility is overshadowed by the cost associated with their production, which often relies on the ability to identify minor antigen-specific cells out of a heterogeneous population. To address concerns with suboptimal methods for screening cells, we have developed a cell-sorting array composed of nanoliter spherical cell culture compartments termed microbubble (MB) wells. We demonstrate a proof-of-concept system for the detection of cell secreted factors from both immortalized cell lines and primary B cell samples. Exploiting the unique ability of the MB well architecture to accumulate cell secreted factors as well as affinity capture coatings, we demonstrate on-chip detection and recovery of antibody-secreting cells for sequencing of immunoglobin genes. Furthermore, rapid image capture and analysis capabilities were developed for the processing of large MB arrays, thus facilitating the ability to conduct high-throughput screening of heterogeneous cell samples faster and more efficiently than ever before. The proof-of-concept assays presented herein lay the groundwork for the progression of MB well arrays as an advanced on-chip cell sorting technology.

Quantitative analysis of spherical microbubble cavity array formation in thermally cured polydimethylsiloxane for use in cell sorting applications

Microbubbles are spherical cavities formed in thermally cured polydimethylsiloxane (PDMS) using the gas expansion molding technique. Microbubble cavity arrays are generated by casting PDMS over a silicon wafer mold containing arrays of deep etched pits. To be useful in various high throughput cell culture and sorting applications it is imperative that uniform micron-sized cavities can be formed over large areas (in(2)). This paper provides an in-depth quantitative analysis of the fabrication parameters that effect the microbubble cavity formation efficiency and size. These include (1) the hydrophobic coating of the mold, (2) the mold pit dimensions, (3) the spatial arrangement of the pit openings, (4) the curing temperature of PDMS pre-polymer, (5) PDMS thickness, and (6) the presence and composition of residual gas in the PDMS pre-polymer mixture. Results suggest that the principles of heterogeneous nucleation and gas diffusion govern microbubble cavity formation, and that surface tension prevents detachment of the vapor bubble that forms in the PDMS over the pit. Paramerters are defined that enable the fabrication of large format arrays with uniform cavity size over 6 in(2) with a coefficient-of-variation <10 %. The architecture of the microbubble cavity is uniquely advantageous for cell culture. Large format arrays provide a highly versatile system that can be adapted for use in various high-throughput cell sorting applications. Herein, we demonstrate the use of microbubble cavity arrays to dissect the cellular heterogeneity that exists in a tumorigenic cutaneous squamous cell carcinoma cell line at the single cell level.

Enriching and characterizing cancer stem cell sub-populations in the WM115 melanoma cell line

Cutaneous melanoma is an increasingly common and potentially lethal malignancy of melanocytes, the melanin producing cells normally located in the basal layer of the skin epidermis. Despite major advances in cancer chemotherapeutics and immunotherapy, the success in treating metastatic melanoma remains poor. The notion that cancer stem cells (CSCs) play a key role in melanoma progression is well received. Therefore, isolating and characterizing CSCs is of critical importance for designing new therapeutic strategies that target this unique tumor initiating cell sub-population. In this work, we present a simple in vitro method, employing cell culture on polydimethylsiloxane (PDMS) and transfer back onto standard tissue culture plate, to enrich a non-adherent spheroid (NA/S) forming and an adherent monolayer (AM) cell sub-populations from the tumorigenic WM115 melanoma cell line. The phenotypes of the morphologically distinct NA/S and AM sub-populations were further characterized by quantifying the expression of stem cell markers, CD20 and CD271. Flow cytometric analysis found 2.32% of the cells in the NA/S sub-population were CD20+ CD271+ whereas only 0.27% of the cells in the AM sub-population were CD20+ CD271+. When the NA/S sub-population was cultured back onto PDMS it resulted in the further enrichment of CD20+ CD271+ cells to 14.7%. We used microbubble arrays to quantify the in vitro clonogenic potential of the NA/S and AM cell sub-populations. Microbubbles are spherical cavities, ∼160 μm in diameter with 60 μm circular openings, formed in PDMS using the gas expansion molding (GEM) process. Cells from each sub-population were seeded, under limiting dilution conditions, onto separate arrays containing 1215 microbubble wells. After five days in culture, wells seeded with 1, 2, 3 and >3 cells per microbubble well were inspected for cell proliferation. The Extreme Limiting Dilutions Analysis (ELDA) determined a ∼58% clonal survival (1 in every 1.72 cells) for the NA/S sub-population and ∼25% clonal survival (1 in every 3.93 cells) for the AM sub-population (= 176, p = 4.41e -40). These findings taken together add to the existing evidence that melanoma cells propagating as non-adherent/spheroids represent a more aggressive phenotype due to the greater presence of tumor initiating cells.

Publisher's Note: “Continuously perfused microbubble array for 3D tumor spheroid model” [Biomicrofluidics 5, 024110 (2011)

This article was originally published online on 3 June 2011 without acknowledgment of the funding from NIH Grant No. {type:entrez-nucleotide,attrs:{text:CA143876,term_id:35040294,term_text:CA143876}}CA143876. AIP apologizes for this error. The article was corrected on 6 June 2011.

Continuously perfused microbubble array for 3D tumor spheroid model

Multi-cellular tumor spheroids (MCTSs) have been established as a 3D physiologically relevant tumor model for drug testing in cancer research. However, it is difficult to control the MCTS testing parameters and the entire process is time-consuming and expensive. To overcome these limitations, we developed a simple microfluidic system using polydimethylsiloxane (PDMS) microbubbles to culture tumor spheroids under physiological flow. The flow characteristics such as streamline directions, shear stress profile, and velocity profile inside the microfluidic system were first examined computationally using a COMSOL simulation. Colo205 tumor spheroids were created by a modified hanging drop method and maintained inside PDMS microbubble cavities in perfusion culture. Cell viability inside the microbubbles was examined by live cell staining and confocal imaging. E-selectin mediated cell sorting of Colo205 and MDA-MB-231 cell lines on functionalized microbubble and PDMS surfaces was achieved. Finally, to validate this microfluidic system for drug screening purposes, the toxicity of the anti-cancer drug, doxorubicin, on Colo205 cells in spheroids was tested and compared to cells in 2D culture. Colo205 spheroids cultured in flow showed a threefold increase in resistance to doxorubicin compared to Colo205 monolayer cells cultured under static conditions, consistent with the resistance observed previously in other MCTS models. The advantages presented by our microfluidic system, such as the ability to control the size uniformity of the spheroids and to perform real-time imaging on cells in the growth platform, show potential for high throughput drug screening development.

Microfabrication of Bubbular Cavities in PDMS for Cell Sorting and Microcell Culture Applications

We describe a novel technique, low surface energy Gas Expansion Molding (GEM), to fabricate microbubble arrays in polydimethylsiloxane (PDMS) which are incorporated into parallel plate flow chambers and tested in cell sorting and microcell culture applications. This architecture confers several operational advantages that distinguish this technology approach from currently used methods. Herein we describe the GEM process and the parameters that are used to control microbubble formation and a Vacuum-Assisted Coating (VAC) process developed to selectively and spatially alter the PDMS surface chemistry in the wells and on the microchannel surface. We describe results from microflow image visualization studies conducted to investigate fluid streams above and within microbubble wells and conclude with a discussion of cell culture studies in PDMS.

Two-dimensional micro-bubble actuator array to enhance the efficiency of molecular beacon based DNA micro-biosensors

Two-dimensional micro-bubble actuator arrays were developed and studied in detail to enhance the hybridization kinetics of a DNA micro-biosensor. The hybridization between a molecular beacon, a kind of oligonucleotide probe, and its complement was investigated in a millimeter-sized PDMS based reaction chamber, where various 2D micro-heater arrays were distributed on the bottom for micro-bubble generation. The hybridization assay without the micro-bubble actuation revealed that the fluorescence increased fast at the beginning and slowed down after that. However, a uniform fluorescence increase was observed when periodic micro-bubble agitation was introduced in the static hybridization solution. A comparison of hybridization assays with and without micro-bubble agitation revealed that the hybridization time could be effectively shortened by 33% with 10 cycles of micro-bubble agitation from a 2 x 1 bubble actuator array, and by 43% with 10 cycles of micro-bubble agitation from a 2 x 2 bubble actuator array.