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 Bubblesâ€“Cell 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>
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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 Bubblesâ€“Cell Interaction and the Resultant Bioeffects at the Single-cell Lev

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&#8211;Cell 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.

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

The overall goal of this experimental procedure is to investigate cavitation induced bioeffects in microfluidic confinement using surface patterning to precisely control the generation of tandem bubbles and the location and shape of the individual target cells. This microfluidic system enables experimentation of this transit cavitation bubbles and cells that is relevant to therapeutic and sound publication and sound operation. The main advantage of this technique comes from the improve the precision from surface patterning.It allows us to study the bioeffects of individual cells and is a high stream loading from reliable bubble-bubble interaction. Visual demonstration of the procedures is critical as the surface patterning and cell preparation in the chip steps are complex involving various techniques and tips. Perform all the microfabrication procedures in a clean room while wearing a clean room suit.Design the area of each gold dot within 25 to 30 square microns, so that it is large enough to absorb laser energy for bubble generation, but small enough to avoid individual cells adhering to it. Clean the glass slide in a chemical hood according to the text protocol, then proceed to the spin coating hood. Program the spin coder to accelerate to 1000 RPM over two seconds and to maintain that speed for five seconds then have it ramp to 3000 RPM over three seconds and maintain that speed for 30 seconds.Next, secure the glass slide to the spin coder using the vacuum. Then, cover the slide with P20 and start the spin cycle. Next, apply NFR negative photo resist using the same cycle.Now, bake the slide on a hot plate at 95 degrees celsius for 60 seconds. Once cooled to room temperature perform the photolithography. Mount the chrome mask onto the mask aligner and make sure that the pattern side is facing down towards the slide.Then, set the photolithography recipe to hard exposure mode with nine seconds of UV exposure and align the glass substrate with the mask. After the UV exposure, bake the slide at 95 degrees celsius for one minute, and then let it cool as before. To develop the pattern on the slide, place it in developer solution for 60 seconds, then wash the slide with distilled water and dry it with nitrogen gas.After confirming the pattern by microscopic inspection bake the slide at 120 degrees celsius for five minutes and let it cool to room temperature. Now, clean the slide with plasma in the reactive ion etching machine for 90 seconds at 500 tor and 100 watts. Using an E-beam evaporator, tape the sample to the plate holder.Program the machine to deposit a 5 nanometer layer of titanium, followed by a 15 nanometer layer of gold. Once that position is complete, ventilate the machine. Next, soak the slide overnight in a beaker of photoresist remover solvent to remove the gold resting on top of the NFR resist.The next day, wash the slide with acetone, followed by IPA and then dry it with nitrogen. Rinse the slide with DI water and dry it again with nitrogen. Now, heat the slide at 115 degrees celsius for five minutes.Then, clean the gold dot pattern slide using an oxygen plasma asher at 100 watts for 90 seconds. Set the area of each fibronectin encoded island to be within 700 to 900 square microns to facilitate adequate hela cell spreading in a square region while minimizing the chances of multiple cells aggregating on the island. Fabrication is much like that of the gold pattern.Spin code the slide as before using S18 positive photoresist and bake on the coding at 115 degrees celsius. Then, perform photolithography, aligning the marks on the mask with those on the substrate. Check the pattern on the central portion to confirm the correct angle and adjust the standoff distance from the gold dots to the H pattern.Run the UV exposure for nine seconds with no post bake. The next difference is that the development step only takes 45 seconds. For the reactive ion etching, set the parimeters to 500 tor 100 watts, and 90 seconds.Then, apply a drop of PLLG peg passivating solution onto a piece of paraffin film and sandwich the solution to the pattern side of the slide. Avoid trapping bubbles. After 45 minutes, remove the slide from the film.Rinse the slide with DI water and then dry it with nitrogen. Next, soak the slide consecutively in photoresist remover 1165. Then 50 percent 1165 in DI water.Then just DI water. During each bath, agitate the solution in an ultrasound bath for 90 seconds. Now, dry the slide on a hot plate, then seal it in a desiccator and store it at 4 degrees celsius.Assemble the slides into a microchannel chip as described in the text protocol. Pay careful attention to shield the patterned area of the glass substrate with the small PDMS slab before using reactive ion etching to remove the PLLG peg from the peripheral area. Retrieve the patterned glass from the RIE machine and remove the small PDMS slab.After treating the microchannel PDMS with a reduced dose of oxygen plasma, align it to the patterned glass substrate under a stereoscope. Bring the micro channel PDMS and the patterned glass substrate in conformal contact. Now, proceed to use the chip.First, prime the chip by flowing PBS down the microchannel for 30 minutes at one microliter per minute. Then infuse the chip with fibronectin solution for 45 minutes at the same flow rate. While waiting, prepare the cells at five million cells per milliliter.Healthy status and high confluencey is critical to this procedure. Later, replace the solution flowing through the chip with PBS and increase the flow rate to ten microliters per minute. Let the PBS flow for five minutes.Next, inject the prepared cells into the chip with a syringe. When one drop has flowed out of the tubing stop the injection and clamp the outlet. Then, place the chip in an incubator for 30 minutes.Later, release the outlet and flush the chip with cell culture medium at ten microliters per minute for five minutes. After five minutes, drop the flow to 0.75 microliters per minute and transfer the chip and pump to an incubator for two hours. After two hours, proceed with tandem bubble generation viewing the chip under an inverted microscope with two pulsed NDI lasers on timing controls directed at the gold dot pattern and with a high speed camera ready to capture the results.For 50 micron bubbles, set the delay between the two lasers to 2.5 microseconds and set their power to ten microjoules. Then, synchronize the high speed camera recording with the lasers and make records at two million frames per second with 200 nanosecond exposures. Thus, the dynamics of bubble expansion, collapse bubble-bubble interaction, and jet formation are recorded.Bubble-bubble interactions and a variety of cavitation induced bioeffects were studied at the single cell level using the described technique, such as the transient interactions of tandem bubbles with the jet formation the visualization of the resultant flow field and the calculation of the jet speed. Directional jetting flow around the tandem bubble is around ten meters per second, is about ten microns wide and is thus capable of producing an impulsive and localized sheer stress and stress gradient onto nearby target cells. Tandem bubble induced cell membrane deformation was also studied.Membrane deformation and recovery are highlighted by the displacement of functionalized beads attached to the leading edge of the cell membrane. From the coordinates of a triad of adjacent beads, the local area strain was calculated. This schematic shows the maximum area change at different locations on the cell surface.The leading edge is primarily stretched, while the trailing edge or lateral sides of the cell are compressed indicating heterogeneity and cell deformation produced by the tandem bubble induced jetting flow. The temporal variation of the area strain at the cell leading edge is comprised of a few rapid oscillations followed by a large and sustained stretch for about 100 microseconds, and a subsequent gradual recovery on a timescale of several milliseconds. After watching this video, you should have a good understanding of how to make a controllable bubble-bubble interaction to study the bioeffects of single cells with controlled shape and location from surface patterning.Following this procedure, other measures like cytoskeleton thinning and the conjugal imaging can be further performed to study cell cytoskeleton rearrangement of the tandem bubble treatment. After its development, this technique will pave the way for researchers in the field of subpretic ultrasound to explore captation induced bioeffects on the single cell level. While attempting this procedure, it is important to remember to maintain a good cell confluency and condition for the success during the cell patterning.Do not forget that working with clean room chemicals and lasers can be extremely hazardous and precautions such as wearing gloves and laser goggles should always be taken while performing this procedure.

Research

JoVE Journal

Bioengineering

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