FAST LOCALIZED SINGLE CELL MEMBRANE PORATION BY BUBBLE-INDUCED JETTING FLOW

Z.G. Li1, C.D. Ohl2, J.B. Zhang3, J. Tsai4 and A.Q. Liu1* 1School of Electrical & Electronic Engineering

2School of Physical and Mathematical Sciences, Nanyang Technological University, SINGAPORE 3Data Storage Institute, A*STAR, SINGAPORE

4Institute of Microelectronics, A*STAR, SINGAPORE ABSTRACT

This paper demonstrates a new method to porate single suspension living cell membrane using cavitation bubble-induced high speed jet flow. A microfluidic chip with an array of single cell trapping structures is designed and fabricated to trap sing cell and induce the asymmetric collapse of the cavitation bubble. Myeloma cells suspended in Trypan blue saline solution is tested and single Myeloma cells can be trapped in the trapping structures. The dynamic process is recorded using a high speed camera. This method has great potential in biomedical applications and easy to be integrated to other microfluidic system. INTRODUCTION

Cavitation usually brings damage to ship propeller blades and hydraulic equipments [1]. However, in recent years, it has been applied to many biological and engineering applications [2], such as kidney stone breakup, and drug delivery using ultrasound [3]. With the development of microfluidics, it has been used to pump fluid [4] or fuse micro-droplets [5] in microfluidic chips. Cavitation bubble has been used to apply poration on suspension cells [6] or single adherent cell [7]. However, the method cannot precisely control the location and the size of the pores on suspension cells’ membranes [6]. The method in ref [7] only can be used on adherent cells. The collapses of cavitation bubble have been studied for hundred years [8]. Different boundary conditions induce different types of collapse for cavitation bubble [9]. If a cavitation bubble collapse close to a solid (rigid) boundary, it collapse asymmetrically and a jet flow towards the solid boundary is developed [2,9]. It has great potential in single cell study to be used as a “fluid needle”.

In this paper, a method is developed to porate single suspension cell membrane using cavitation bubble-induced jet flow. A microfluidic chip with an array of single cell trapping geometries are designed and fabricated. A cavitation bubble is created close to a trapped cell, and the boundary conditions introduce the asymmetric collapse of the bubble. A jet flow towards the trapped cell and two vortices is formed to porate and stretch the cell, respectively. Using this method, single suspension cell membrane can be porated using jet flows with precise direction and strength.

DESIGN

The mechanism of single cell poration using jet flow is shown in Fig. 1. Cells suspended in Trypan blue solution is injected into the microfluidic chip and trapped by the trapping geometries one by one. A cavitation bubble is

created using a pulse laser system close to the trapped cell (Fig. 1(a)). After the bubble expands to its maximum (Fig. 1(b)), the bubble begins to collapse in an asymmetric type due to the boundary conditions created by the trapping geometries (Fig. 1(c)). Two vortices rotating in opposite directions and one jet flow towards the trapped cell is induced by the asymmetric collapse. The cell is deformed and porated by the jet flow and stretched along the direction perpendicular to the direction of the jet flow by two vortices (Fig. 1(d-e)). In longer time scale, the deformed cell restores its original shape by the elastic force and the Trypan blue molecules gradually diffuse to the cell cytosol (Fig. 1(f)).

Figure 1: Schematic of single cell membrane poration by cavitation bubble-induced jetting flow.A cavitation bubble is created close to the trapped cell (a) and the bubble expands to the maximum (b). Then, the bubble collapses asymmetrically since the solid boundary condition of the trapping structure (c). A jet flow is induced since the asymmetric collapse (d-e) and the trapped cell is deformed and porated by the jet flow (e). The trypan blue molecules diffuse into the porated cell (f) MATERIALS AND METHODS Microfluidic Chip

A microfluidic chip with an array of cell trapping structures is designed and fabricated [5]. Single trapping structure consists of one equilateral triangular structure and one microchannel as shown in Fig 2. The chip has one inlet and one outlet. For trapping structure, the length of the side of the equilateral triangle w1 is 20 µm and the widths of the micro apertures w2 vary from 5 µm, 7.5 µm to 10 µm. All channels have a depth of 27 µm.

The microfluidic chip is fabricated using standard

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978-1-4673-0325-5/12/$31.00 ©2012 IEEE 819 MEMS 2012, Paris, FRANCE, 29 January - 2 February 2012

Pumping Fluid with Suspended Myeloma Cells

An Array of Single Cell Trapping Structures

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Figure 2: Schematic of the microfluidic chip with an array of sing suspension cell trapping structures (not scale).

softlithography techniques. The master is fabricated using SU8 photoresist (SU8-10, MicroChem) on a silicon wafer substrate. After spinning (CEE 200, Brewer Science), prebaking, exposure to UV light (506, OAI), postbaking and developing, the master with the thickness 27 µm are fabricated. A mixture of poly(dimethylsiloxane) (PDMS, Sylgard 184, Dow Corning) pre-polymer and curing agent (10:1) is poured over the master. After degassed, 2 hours baking, peeled off, and holes punching (0.75 µm, Harris unicore), the PDMS layer with micro-structures is bonded with a glass substrate using a corana surface treater (BD-25, Electro-Technic Products, USA) to form a sealed microfluidic chip.

Cell Trapping

Myeloma cells with 105 cells/ml concentration suspended in the 0.4% saline Trypan blue solution (T5184, Sigma-Aldrich) is pumped into the microfluidic chip, which flowing through trapping geometries will be trapped. Trypan blue enhanced laser absorption to facilitate cavitation generation and it also used to label the poration, since the Trypan blue molecules will diffuse into the cell once the cell is porated. The inset in Fig 3 is the image of single cell trapping structure with a trapped Myeloma cell. The scale bar is 10 µm. The depth of whole chip is 27 µm.

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Number of Cell Trapped Figure 3: The statistical result of cell trapping. The inset image shows one single trapped myeloma cell. The scale bar is 20 µm.

The statistical results of the cell trapping is shown in Fig 3. In total 94 testing trapping structures, 20 % trapping structures is null, up to 50 % trap single cell and 30 % trap two or more cells. This trapping efficiency is good enough for the poration experiments.

RESULTS AND DISCUSSIONS Microjets An optical setup is used to generate cavitation bubble. A single pulse from an Nd:YAG laser at the wavelength of 532 nm with a duration of 6 ns is used to create cavitation bubble. Images are recorded with a high-speed camera (Photron SA-1) at 552 000 frames per second with an exposure time 1 µs. The dynamics of single bubble with/without a rigid boundary are shown in Fig. 4(a) and (b), respectively. A cavitation bubble is created at the location close to a rigid boundary. It induced the asymmetrical growth and collapse of the cavitation bubble. After the collapse, a jet towards the rigid boundary is formed [Fig. 4(b), 10-14µs]. The jet project the residue of the cavitation bubble to the boundary. The formation of jets is not observed during the symmetric growth and collapse of a single bubble without rigid boundary as shown in Fig. 4(c). The residue of the bubble always stays at the same location after the collapse of the bubble.

Figure 4: Dynamics of bubble close to a rigid boundary. (a) Dynamics of single bubble close to a rigid boundary. (b) Dynamics of single bubble without any boundary. The rigid boundary induces the asymmetrical growth and collapse of the single bubble and a jet towards the rigid boundary after collapse. The scale bar is 25 µm. Particle Tracking

For the visualization of the flow field of the generation of the microjet, 2 µm polystyrene beads (1×106 beads/ml, Duke Scientific) added in the refill ink for inkjet printer (red, Maxtec) with dynamic viscosity 2 mPa·s are used as tracers. When a caivtion bubble (60 µm in diameter) grows and collapses asymmetrically, a pair of vortices and a microjet is observed. A Matlab code is developed to track the motion of microbeads and calculate the velocity field. One vortex rotates clockwise and the other counterclockwise as in Fig. 5, with a maximum vorticity is up to 30 000 s-1.

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Figure 5: Particle tracking results to show the jet flow and two vortices. The scale bar is 50 µm.

Single Cell Poration

The selected high-speed images of the dynamics process of the poration of single Myeloma cell are shown in Fig. 6. A Myeloma cell is trapped first. A cavitation bubble close to the cell is created at t = 0 µs. The trapped cell is pushed towards the trapping structure during the bubble expansion. The bubble contracts at t = 4 µs and then collapses. The membrane of the trapped cell is deformed and porated at t = 6 µs by the high speed jet flow due to the asymmetric collapse of the cavitation bubble. Then, the cell’s membrane begins to restore since elastic force of the cell membrane and the process is lasting for approximately 8 ms.

Figure 6: Single-cell membrane poration dynamic process. A myeloma cell is trapped, and a bubble close to the cell is created at 0 µs. The bubble starts to collapse at t = 4 µs and the cell is deformed and porated at t = 6 µs. The scale bar is 10 µm. Trypan Blue Diffusion

The selected high speed images of the Trypan blue uptake process are shown in Fig. 7. The Trypan blue diffuses into the cell cytosol in approximately 28 s. The direction of the diffusion of the Trypan blue is from top to the bottom, which indicates that the cell membrane is porated at the top of the cell.

Figure 7: The Trypan blue uptake process of the porated cell.

Statistics experimental results in Fig.8 show the cell

membrane poration strongly depends on the stand-off distances. The diffusion distance after 3 seconds the bubble

presence is measured for three group experiments with different stand-off distance. Statistically significant increase of the average diffusion distance from 6.9 µm, 8.9 µm and 10.1 µm is observed when the stand-off distance decreases from 25 µm, 20 µm to 15 µm. When the stand-off distance increase to 30 µm, the target cells are not porated even 30 seconds after the bubble created. The error bars account for the fluctuation of the diffusion distance for 10 repeated experiments in one group. These statistics results indicate that smaller stand-off distance under same laser energy (same bubble size), the jet creates bigger pore at the membrane of the cell.

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Stand-off Distance (μm) Figure 8: The statistical result of the diffusion distance of Trypan blue at 3s after the presence of the bubble under different stand-off distances.

CONCLUSIONS

In conclusion, a method for single suspension cell membrane poration is developed using cavitation bubble-induced microjets. A microfluidic chips with an array of single cell trapping structuress is designed and fabricated using standard soft-lithography technology. Myeloma cells suspended in Traypan blue saline solution are flushed into the chip and trapped one by one. A cavitation bubble is created at the location close to one single trapped cell. Due to the boundary conditions formed by the tapping structure, the bubble grows and collapses asymmetrically and induces one microjet towards the trapped cell (the boundary) and two vortices rotating in opposite directions. The cell is deformed and porated by the mircojet and Trypan blue molecules diffusing to the cytosol of the cell indicates the poration is realized by the microjet. The statistics experimental results show that with the increase of the stand-off distance, the diffusion distance after cells’ poration decrease. This method provides new solution to single suspension cell membrane poration with precisely controlling of the direction and strength. It is also easily to be integrated into other microfluidic systems. ACKNOWLEDGEMENTS

The work is supported by the Environmental and Water Industry Development (EWI) Council of Singapore, research project (Grant No. MEWR C651/06/171) and the Ministry of Education (MOE) Singapore, through the Tier 2 project (Grant No. T208A1238).

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[6] S. L. Gac, E. Zwaan, A. B. D. Berg, and C. D. Ohl, "Sonoporation of suspension cells with a single cavitation bubble in a microfluidic confinement, " Lab Chip, vol. 7, pp. 1666 – 1672, 2007.

[7] G. N. Sankin, F. Yuan, and P. Zhong, "Pulsating Tandem Microbubble for Localized and Directional Single-Cell Membrane Poration," Phys. Rev. Lett, vol. 105, 078101, 2010.

[8] L. Rayleigh, ""On the pressure developed in a liquid during the collapse of a spherical cavity," Philos. Mag., vol. 34, pp. 94 – 98 1917.

[9] J. R. Blake, and D. C. Gibson, “Gavitation bubbles near boundaries,” Annu. Rev. Fluid Mech., vol. 19, pp. 99-123, 1987

CONTACT

*A. Q. Liu, Tel: +65-6790 4336; Eaqliu@ntu.edu.sg

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