Name: a microfluidic system with surface patterning for investigating cavitation bubble(s)&#8211;cell interaction and the resultant bioeffects at the single-cell level
Uploaded: 2017-01-10T15:00:00
Description: Watch this Scientific Journal Video about A Microfluidic System with Surface Patterning for Investigating Cavitation BubblesÃ¢â‚¬â€œCell Interaction and the Resultant Bioeffects at the Single-cell Lev

We would like to acknowledge the use of the clean room facility SMIF at Duke University. We also want to thank Hao Qiang for his assistance in measuring the jet velocity. The authors thank Todd Rumbaugh of Hadland Imaging for providing the Shimadzu HPV-X camera used in this study.The work was funded in part by NIH through grants 5R03EB017886-02 and 4R37DK052985-20.

1. Microfabrication
NOTE: All the microfabrication procedures are performed in a cleanroom. A chrome mask is designed prior to the microfabrication, see Figure 2.
<img alt="Figure 2" src="/files/ftp_upload/55106/55106fig2.jpg" /> 
Figure 2: Schematic of the channel design in the microfluidic chip and the dimensions of the working units. a) Mask design of the aligned PDMS microchannels (green) and ...

<ol>
	<li>Wang, D., Bodovitz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+analysis:+the+new+frontier+in+'omics.">Single cell analysis: the new frontier in 'omics.</a> Trends Biotechnol. 28 (6), 281-290 (2010).</li><li>Weaver, W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+high-throughput+single-cell+microtechnologies.">Advances in high-throughput single-cell microtechnologies.</a> Curr Opin Biotechnol. 25, 114-123 (2014).</li><li>Gossett, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+stretching+of+single+cells+for+large+population+mechanical+phenotyping.">Hydrodynamic stretching of single cells for large population mechanical phenotyping.</a> Proc Natl Acad Sci USA. 109 (20), 7630-7635 (2012).</li><li>Spiller, D. G., Wood, C. D., Rand, D. A., White, M. R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+single-cell+dynamics.">Measurement of single-cell dynamics.</a> Nature. 465 (7299), 736-745 (2010).</li><li>Lecault, V., White, A. K., Singhal, A., Hansen, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+single+cell+analysis:+from+promise+to+practice.">Microfluidic single cell analysis: from promise to practice.</a> Curr Opin Chem Biol. 16 (3-4), 381-390 (2012).</li><li>Kennedy, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-intensity+focused+ultrasound+in+the+treatment+of+solid+tumours.">High-intensity focused ultrasound in the treatment of solid tumours.</a> Nat Rev Cancer. 5 (4), 321-327 (2005).</li><li>Zhu, S., Cocks, F. H., Preminger, G. M., Zhong, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+stress+waves+and+cavitation+in+stone+comminution+in+shock+wave+lithotripsy.">The role of stress waves and cavitation in stone comminution in shock wave lithotripsy.</a> Ultrasound Med Biol. 28 (5), 661-671 (2002).</li><li>Fan, Z., Liu, H., Mayer, M., Deng, C. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporally+controlled+single+cell+sonoporation.">Spatiotemporally controlled single cell sonoporation.</a> Proc Natl Acad Sci U S A. 109 (41), 16486-16491 (2012).</li><li>Itah, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+cavitation+kills+prostate+cells+and+ablates+benign+prostatic+hyperplasia+tissue.">Hydrodynamic cavitation kills prostate cells and ablates benign prostatic hyperplasia tissue.</a> Exp Biol Med. 238 (11), 1242-1250 (2013).</li><li>Kosar, A., Sesen, M., Oral, O., Itah, Z., Gozuacik, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bubbly+cavitating+flow+generation+and+investigation+of+its+erosional+nature+for+biomedical+applications.">Bubbly cavitating flow generation and investigation of its erosional nature for biomedical applications.</a> IEEE Trans Biomed Eng. 58 (5), 1337-1346 (2011).</li><li>Li, Z. G., Liu, A. Q., Klaseboer, E., Zhang, J. B., Ohl, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+membrane+poration+by+bubble-induced+microjets+in+a+microfluidic+chip.">Single cell membrane poration by bubble-induced microjets in a microfluidic chip.</a> Lab Chip. 13 (6), 1144-1150 (2013).</li><li>Li, F. F., Chan, C. U., Ohl, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yield+Strength+of+Human+Erythrocyte+Membranes+to+Impulsive+Stretching.">Yield Strength of Human Erythrocyte Membranes to Impulsive Stretching.</a> Biophys J. 105 (4), 872-879 (2013).</li><li>Li, F., M, M., Ohl, C. .. D. .. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear+stress+induced+stretching+of+red+blood+cells+by+oscillating+bubbles+within+a+narrow+gap.">Shear stress induced stretching of red blood cells by oscillating bubbles within a narrow gap.</a> Bull Am Phys Soc. 58, (2013).</li><li>Sankin, G. N., Yuan, F., Zhong, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsating+tandem+microbubble+for+localized+and+directional+single-cell+membrane+poration.">Pulsating tandem microbubble for localized and directional single-cell membrane poration.</a> Phys. Rev. Lett. 105 (7), 078101 (2010).</li><li>Fan, Q., Hu, W., Ohta, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-induced+microbubble+poration+of+localized+single+cells.">Laser-induced microbubble poration of localized single cells.</a> Lab Chip. 14 (9), 1572-1578 (2014).</li><li>Chen, C. S., Mrksich, M., Huang, S., Whitesides, G. M., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometric+control+of+cell+life+and+death.">Geometric control of cell life and death.</a> Science. 276 (5317), 1425-1428 (1997).</li><li>Yuan, F., Sankin, G., Zhong, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+tandem+bubble+interaction+in+a+microfluidic+channel.">Dynamics of tandem bubble interaction in a microfluidic channel.</a> J Acoust Soc Am. 130 (5), 3339-3346 (2011).</li><li>Yang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analysis of Tandem Bubble Interaction and Jet Formation in a Microfluidic Channel. , (2013).</li><li>Simon, S. I., Schmid-Schonbein, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+strains+and+strain+rates+in+motile+polymorphonuclear+leukocytes.">Cytoplasmic strains and strain rates in motile polymorphonuclear leukocytes.</a> Biophys J. 58 (2), 319-332 (1990).</li><li>Barbee, K. A., Macarak, E. J., Thibault, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain+measurements+in+cultured+vascular+smooth+muscle+cells+subjected+to+mechanical+deformation.">Strain measurements in cultured vascular smooth muscle cells subjected to mechanical deformation.</a> Ann Biomed Eng. 22 (1), 14-22 (1994).</li><li>Yuan, F., Yang, C., Zhong, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+membrane+deformation+and+bioeffects+produced+by+tandem+bubble-induced+jetting+flow.">Cell membrane deformation and bioeffects produced by tandem bubble-induced jetting flow.</a> Proc. Natl. Acad. Sci. U.S.A. 112 (51), E7039-E7047 (2015).</li><li>Yin, H., Marshall, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+for+single+cell+analysis.">Microfluidics for single cell analysis.</a> Curr Opin Biotechnol. 23 (1), 110-119 (2012).</li><li>Tay, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+NF-kappa+B+dynamics+reveal+digital+activation+and+analogue+information+processing.">Single-cell NF-kappa B dynamics reveal digital activation and analogue information processing.</a> Nature. 466 (7303), 267-271 (2010).</li><li>Rand, R. P., Burton, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+Properties+of+the+Red+Cell+Membrane:+I.+Membrane+Stiffness+and+Intracellular+Pressure.">Mechanical Properties of the Red Cell Membrane: I. Membrane Stiffness and Intracellular Pressure.</a> Biophys J. 4 (2), 115-135 (1964).</li><li>Lim, C. T., Dao, M., Suresh, S., Sow, C. H., Chew, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+deformation+of+living+cells+using+laser+traps.">Large deformation of living cells using laser traps.</a> Acta Mater. 52 (7), 1837-1845 (2004).</li><li>Puig-de-Morales-Marinkovic, M., Turner, K. T., Butler, J. P., Fredberg, J. J., Suresh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscoelasticity+of+the+human+red+blood+cell.">Viscoelasticity of the human red blood cell.</a> Am J Physiol Cell Physiol. 293 (2), C597-C605 (2007).</li><li>Kudo, N., Okada, K., Yamamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonoporation+by+single-shot+pulsed+ultrasound+with+microbubbles+adjacent+to+cells.">Sonoporation by single-shot pulsed ultrasound with microbubbles adjacent to cells.</a> Biophys J. 96 (12), 4866-4876 (2009).</li><li>van Wamel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vibrating+microbubbles+poking+individual+cells:+Drug+transfer+into+cells+via+sonoporation.">Vibrating microbubbles poking individual cells: Drug transfer into cells via sonoporation.</a> J Control Release. 112 (2), 149-155 (2006).</li><li>Hu, Y., Wan, J. M., Yu, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+perforation+and+recovery+dynamics+in+microbubble-mediated+sonoporation.">Membrane perforation and recovery dynamics in microbubble-mediated sonoporation.</a> Ultrasound Med Biol. 39 (12), 2393-2405 (2013).</li><li>Dijkink, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+cavitation-cell+interaction:+trans-membrane+transport+and+viability+studies.">Controlled cavitation-cell interaction: trans-membrane transport and viability studies.</a> Phys Med Biol. 53 (2), 375-390 (2008).</li><li>Rau, K. R., Quinto-Su, P. A., Hellman, A. N., Venugopalan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsed+laser+microbeam-induced+cell+lysis:+Time-resolved+imaging+and+analysis+of+hydrodynamic+effects.">Pulsed laser microbeam-induced cell lysis: Time-resolved imaging and analysis of hydrodynamic effects.</a> Biophys J. 91 (1), 317-329 (2006).</li></ol>

Single-cell analysis, in combination with live-cell imaging, has greatly enhanced our understanding of the dynamic and often variable processes in individual cells, such as phenotype development and immune response23. In contrast to the conventional cell culture in dishes or flasks, microfluidic systems enable precise control of the microenvironment, down to the single-cell level, in real time. Consequently, advances in microfluidic technology and techniques have largely improved the throughput and reproducibi...

There is a growing recognition that cellular heterogeneity, arising from the stochastic expression of genes, proteins, and metabolites, exists within a large cell population and serves as a fundamental principle in biology to allow for cell adaptation and evolution1. Therefore, it is often inaccurate and unreliable to use population-based bulk measurements to understand the function of individual cells and their interactions. Developing new technologies for single-cell analysis is therefore of high interest in biological and pharmacological research, and can be used, for example, to better understand the key signaling pathways and processes in stem cell biology and cancer therapy2-4. In recent years, the emergence of microfluidic platforms has greatly facilitated single-cell analysis, where the positioning, treatment, and observation of the response from individual cells have been performed with novel analytical strategies5.
Cavitation plays an important role in a diverse range of biomedical applications, including the treatment of cancers by high-intensity focused ultrasound (HIFU)6, the non-invasive fragmentation of kidney stones by shock wave lithotripsy (SWL)7, drug or gene delivery by sonoporation8, and the recently reported destruction of cells or tissues by hydrodynamic bubble cavitation9,10. Despite this, the dynamic processes of cavitation bubble(s) interactions with biological tissue and cells have not been well understood. This is due to the randomness in cavitation initiation and bubble dynamics produced by ultrasound, shock waves, and local hydraulic pressure; furthermore, there is a lack of enabling techniques to resolve the inherently complex and fast responses of biological cells, especially at the single-cell level.
Because of these challenges, it is not surprising that very few studies have been reported to investigate bubble-cell interactions under well-controlled experimental conditions. For example, membrane poration of individual cells trapped in suspension11 and the impulsive large deformation of human red blood cells12 have been demonstrated using laser-generated single bubbles in microfluidic channels. The latter technique, however, can only produce very small deformation in eukaryotic cells because of the presence of the nucleus13. Moreover, it is difficult to monitor downstream bioeffects when treating cells in suspension. In other studies, ultrasound excitation of a cell-bound microbubble (or ultrasound contrast agent) for producing membrane poration and/or intracellular calcium responses in single adherent cells has been reported8. Membrane poration of single adherent cells can also be produced by using laser-generated tandem bubbles in a thin liquid layer containing light-absorbing Trypan blue solution14, or by an oscillating gas bubble generated by microsecond laser pulses irradiating through an optically absorbing substrate in microchambers15. When compared, the optically absorbing substrate has an advantage over the laser-absorbing Trypan blue solution because the latter is toxic to cells. More importantly, laser-generated bubbles are more controllable in terms of bubble size and location than acoustically excited bubbles. Nevertheless, in all these previous studies, the cell shape, orientation, and adhesion conditions were not controlled, which may substantially influence the cell response and bioeffects produced by mechanical stresses16.
To overcome these drawbacks in previous studies, we have recently developed an experimental system for bubble generation, cell patterning, bubble-bubble-cell interactions, and real-time bioassays of cell response in a microfluidic chip constructed by using a unique combination of microfabrication techniques. Three main features that distinguish our experimental system from others in the field are: 1) the patterning of micron-sized gold dots on the glass substrate to enable localized laser absorption for bubble generation17; 2) the patterning of micron-sized islands of extracellular matrix (ECM) for cell adhesion on the same substrate to control both the location and geometry of individual cells; and 3) the compression of the dimension of the bubble-bubble-cell interaction domain from 3D to a quasi-2D space to facilitate in-plane visualization of bubble-bubble interactions, jetting flow fields, cell deformation, and bioeffects, all captured in one streamlined imaging sequence (Figure 1d).
<img alt="Figure 1" src="/files/ftp_upload/55106/55106fig1.jpg" /> 
Figure 1: The microfluidic chip and schematics of different assays. a) An assembled microfluidic chip with channels filled with blue ink for visualization. b) A region inside the microfluidic chip with patterned cells and gold dots (the distance between the two gold dots in proximity is 40 &#181;m). Many pairs of working units can be arranged in a channel. c) Close-up image of a single working unit consisting of a pair of gold dots and a HeLa cell adhered to the cell-patterning region. d) Schematic of the device operation. A single cell adheres to and spreads on the &#34;H&#34;-shaped island coated with fibronectin. A pair of cavitation bubbles (tandem bubble) with anti-phase oscillation are produced by illuminating pulsed laser beams on the gold dots (see Figure 4a), leading to the generation of a fast and localized jet moving towards the target cell nearby. The cell may be deformed, porated for macromolecular uptake, and/or stimulated with a calcium response, depending upon the standoff distance (Sd) of the cell to the tandem bubble. <a href="http://cloudflare.jove.com/files/ftp_upload/55106/55106fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This platform can be further combined with fluorescence assays and functionalized beads attached to the cell surface for cavitation-induced bioeffects. In particular, this platform opens the way for reliable and quantifiable assays at the single-cell level. Up to now, we have used the device for the analysis of tandem bubble-induced cell membrane deformation, cell poration and intracellular uptake, viability, apoptosis, and intracellular calcium response. In the following protocol, we describe the process of chip fabrication and the procedure for analyzing the various bioeffects mentioned above. Moreover, the operations of the chip are also described.

<table border="0" cellpadding="0" cellspacing="0" width="678">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Reagent/Materials</td>
			<td style="width:171px;"></td>
			<td style="width:155px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">75x38mm Plain Microscope Slides</td>
			<td style="width:171px;">Corning</td>
			<td style="width:155px;">2947-75X38</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Acetone</td>
			<td style="width:171px;">Sigma Aldrich, Co.</td>
			<td style="width:155px;">320110</td>
			<td style="width:179px;">ACS reagent, &#8805;99.5%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Isopropyl alcohol</td>
			<td style="width:171px;">Sigma Aldrich, Co.</td>
			<td style="width:155px;">W292907</td>
			<td style="width:179px;">&#8805;99.7%, FCC, FG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Sulfuric acid</td>
			<td style="width:171px;">Sigma Aldrich, Co.</td>
			<td style="width:155px;">320501</td>
			<td style="width:179px;">ACS reagent, 95.0-98.0%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Hydrogen peroxide</td>
			<td style="width:171px;">Sigma Aldrich, Co.</td>
			<td style="width:155px;">216763</td>
			<td style="width:179px;">30 wt.% in H2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Primer P-20</td>
			<td style="width:171px;">Microchem</td>
			<td style="width:155px;">MCC Primer 80/20</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">NFR photoresist</td>
			<td style="width:171px;">JSR</td>
			<td style="width:155px;">NFR016D2</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Photomask</td>
			<td style="width:171px;">Photoplotstore</td>
			<td style="width:155px;">N/A</td>
			<td style="width:179px;">4x4 Direct write mask</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">MF-319 Developer</td>
			<td style="width:171px;">Shipley (Rohm and Haas)</td>
			<td style="width:155px;">Microposit MF-319</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">1165 Photoresist Remover</td>
			<td style="width:171px;">Dow Chemical, Co.</td>
			<td style="width:155px;">DEM-10018073</td>
			<td style="width:179px;">1-methyl-2-pyrrolidinone based</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">S1813 photoresist</td>
			<td style="width:171px;">Shipley (Rohm and Haas)</td>
			<td style="width:155px;">S1813</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">PLL-g-PEG</td>
			<td style="width:171px;">SuSoS</td>
			<td style="width:155px;">PLL(20)-g[3.5]- PEG(2)</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">HEPES</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">15630080</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Paraffin film</td>
			<td style="width:171px;">HACH</td>
			<td style="width:155px;">251764</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">SU-2025 photoresist</td>
			<td style="width:171px;">Microchem</td>
			<td style="width:155px;">SU-2025</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">PDMS</td>
			<td style="width:171px;">Dow Corning</td>
			<td style="width:155px;">184 SIL ELAST KIT 0.5KG</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Microbore Tubing</td>
			<td style="width:171px;">Saint-Gobain PPL Corp.</td>
			<td style="width:155px;">S-54-HL</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:175px;">Metal pins</td>
			<td style="width:171px;">New England Small Tube</td>
			<td style="width:155px;">NE-1300-01</td>
			<td style="width:179px;">&#160;Cut Tube (straight), 0.025&#8221; OD x 0.017&#8221; ID x 0.50&#8221; Long</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">HeLa cells</td>
			<td style="width:171px;">Duke Cell Culture Facility</td>
			<td style="width:155px;">(307-CCL-2) HeLa, p.148</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">DPBS(1X) buffer</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">14190144</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">DMEM culture medium</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">11995065</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Fibronectin Bovine Protein, Plasma</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">33010018</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">0.25% Trypsin-EDTA (1X)</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">25200056</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Propidium Iodide</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">P21493</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Carboxylate Microspheres 1.00&#956;m</td>
			<td style="width:171px;">Polysciences, Inc</td>
			<td style="width:155px;">08226-15</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Carboxylate Microspheres 2.00&#956;m</td>
			<td style="width:171px;">Polysciences, Inc</td>
			<td style="width:155px;">18327-10</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:175px;">EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride)</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">22980</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:175px;">Sulfo-NHS</td>
			<td style="width:171px;">Sulfo-NHS (N-hydroxysulfosuccinimide)</td>
			<td style="width:155px;">24510</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Peptite-2000</td>
			<td style="width:171px;">Advanced BioMatrix</td>
			<td style="width:155px;">5020-5MG</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">FITC Annexin V</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">A13199</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Fura-2, AM</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">F1221</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">DMSO</td>
			<td style="width:171px;">Sigma Aldrich, Co.</td>
			<td style="width:155px;">D2650</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">F-127</td>
			<td style="width:171px;">invitrogen</td>
			<td style="width:155px;">P6866</td>
			<td style="width:179px;">0.2 &#181;m filtered (10% Solution in Water)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Reduced serum media&#160;</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">11058021</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Name</td>
			<td style="width:171px;">Company</td>
			<td style="width:155px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:175px;">Plasma asher</td>
			<td style="width:171px;">Emitech</td>
			<td style="width:155px;">K-1050X</td>
			<td style="width:179px;">O2 / Ar plasma ashing of photoresist and other organic materials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Mask aligner</td>
			<td style="width:171px;">SUSS MicroTec&#160;</td>
			<td style="width:155px;">Karl Suss MA6/BA6</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">E-beam evaporator</td>
			<td style="width:171px;">CHA Industries</td>
			<td style="width:155px;">CHA Industries Solution E-Beam</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">RIE</td>
			<td style="width:171px;">Trion Technology&#160;</td>
			<td style="width:155px;">Trion Technology Phantom II</td>
			<td style="width:179px;">(oxide/ nitride/ polymer) etching</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Stereoscope</td>
			<td style="width:171px;">AmScope</td>
			<td style="width:155px;">American Scope SM-4TZ-FRL</td>
			<td style="width:179px;">Stereo Microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Syringe pump</td>
			<td style="width:171px;">Chemyx Inc</td>
			<td style="width:155px;">NanoJet</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:175px;">Cell culture incubator</td>
			<td style="width:171px;">NuAire</td>
			<td style="width:155px;">AutoFlow NU-8500 Water Jacket CO2 Incubator</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Biological Safety Cabinets</td>
			<td style="width:171px;">NuAire</td>
			<td style="width:155px;">NU-425-400</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Water bath</td>
			<td style="width:171px;">VWR</td>
			<td style="width:155px;">1122s</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Centrifuge</td>
			<td style="width:171px;">IEC</td>
			<td style="width:155px;">Centra CL2</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Microscope</td>
			<td style="width:171px;">Zeiss</td>
			<td style="width:155px;">Axio Observer Z1</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Nd:YAG laser&#160; (laser 1)</td>
			<td style="width:171px;">New Wave Research</td>
			<td style="width:155px;">Tempest</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Nd:YAG laser&#160; (laser 2)</td>
			<td style="width:171px;">New Wave Research</td>
			<td style="width:155px;">Orion</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Delay generator</td>
			<td style="width:171px;">Berkeley Nucleonics&#160;</td>
			<td style="width:155px;">BNC 565-8c</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Flash lamp</td>
			<td style="width:171px;">Dyna-Lite</td>
			<td style="width:155px;">ML1000 fiber-coupled flashtube</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">high speed camera</td>
			<td style="width:171px;">DRS Hadland</td>
			<td style="width:155px;">&#160;Imacon 200</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">high speed&#160; camera</td>
			<td style="width:171px;">Shimadzu</td>
			<td style="width:155px;">HPV-X</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">high speed camera</td>
			<td style="width:171px;">Vision Research</td>
			<td style="width:155px;">Phantom V7.3</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">PIV software</td>
			<td style="width:171px;">LaVision</td>
			<td style="width:155px;">DaVis 7.2</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">camera</td>
			<td style="width:171px;">Zeiss</td>
			<td style="width:155px;">AxioCam MRc 5</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">software</td>
			<td style="width:171px;">Zeiss</td>
			<td style="width:155px;">AxioVision</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">PTI system</td>
			<td style="width:171px;">Horiba</td>
			<td style="width:155px;">S/N: 1705 RAM-X</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">EasyRatio software</td>
			<td style="width:171px;">Horiba</td>
			<td style="width:155px;">Easy Ratio Pro 2</td>
			<td style="width:179px;">version 2.3.125.86</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">63&#215; objective</td>
			<td style="width:171px;">Zeiss</td>
			<td style="width:155px;">LD Plan Neofluar</td>
			<td style="width:179px;"></td>
		</tr>
	</tbody>
</table>

a microfluidic system with surface patterning for investigating cavitation bubble(s)&#8211;cell interaction and the resultant bioeffects at the single-cell level

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass substrate, that precisely controls the generation of tandem bubbles and individual cells patterned nearby with well-defined locations and shapes. We then demonstrate the generation of tandem bubbles by using two pulsed lasers illuminating a pair of gold dots with a few-microsecond time delay. We visualize the bubble-bubble interaction and jet formation by high-speed imaging and characterize the resultant flow field using particle image velocimetry (PIV). Finally, we present some applications of this technique for single cell analysis, including cell membrane poration with macromolecule uptake, localized membrane deformation determined by the displacements of attached integrin-binding beads, and intracellular calcium response from ratiometric imaging. Our results show that a fast and directional jetting flow is produced by the tandem bubble interaction, which can impose a highly localized shear stress on the surface of a cell grown in close proximity. Furthermore, different bioeffects can be induced by altering the strength of the jetting flow by adjusting the standoff distance from the cell to the tandem bubbles.

The microfluidic platform described in this work can be used to investigate bubble-bubble interactions and to analyze a variety of cavitation-induced bioeffects at the single-cell level. Here, we present several examples to demonstrate a variety of experimental studies and bioassays that can be performed in our experimental system. We will first illustrate the transient interactions of tandem bubbles with the jet formation, the visualization of the resultant flow field, and the calculatio...

Watch this Scientific Journal Video about A Microfluidic System with Surface Patterning for Investigating Cavitation BubblesÃ¢â‚¬â€œCell Interaction and the Resultant Bioeffects at the Single-cell Lev

A Microfluidic System with Surface Patterning for Investigating Cavitation BubblesÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œCell Interaction and the Resultant Bioeffects at the Single-cell Level

A microfluidic chip was fabricated to produce pairs of gold dots for tandem bubble generation and fibronectin-coated islands for single-cell patterning nearby. The resultant flow field was characterized by particle image velocimetry and was employed to study various bioeffects, including cell membrane poration, membrane deformation, and intracellular calcium response.

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass ...

Research

JoVE Journal

Bioengineering

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Three-Dimensionally Printed Microfluidic Cross-flow System for Ultrafiltration/Nanofiltration Membrane Performance Testing

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...