A SINGLE-CELL MEMBRANE DYNAMIC FROM PORATION TO RESTORATION BY BUBBLE-INDUCED JETTING FLOW

Z. G. Li1, K. Q. Luo2, C. D. Ohl3, J. B. Zhang4 and A. Q. Liu1* 1School of Electrical & Electronic Engineering

2School of Chemical and Biomedical Engineering 3School of Physical and Mathematical Sciences, Nanyang Technological University, SINGAPORE 637371

4Data Storage Institute, 5 Engineering Drive I, SINGAPORE 117608 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. KEYWORDS: Single cell membrane poration, Cavitation bubble, Jetting flow

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.

WORKING PRINCIPLE

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

(a) (b) (c) (d) (e) (f)

Bubble

Cell

BubbleExpansion

BubbleCollapse

Jet Formation Jet Formation &Two Vortices

Cell Restoring& Trypan Blue

Upatake

Figure 1: The mechanism of single cell membrane poration using cavitation bubble-induced jet flow. (a) A cavitation bubble is created close to the trapped cell and (b) the bubble expands to the maximum. (c) The bubble collapses asymmetrically since the solid boundary condition of the trapping structure. (d) A jet flow is induced since the asymmetric collapse and (e) the trapped cell is deformed and porated by the jet flow. (f) The Trypan blue molecules diffuse into the porated cell.

978-0-9798064-4-5/µTAS 2011/$20©11CBMS-0001 94 15th International Conference onMiniaturized Systems for Chemistry and Life Sciences

October 2-6, 2011, Seattle, Washington, USA

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)). EXPERIMENTAL RESULTS AND DISCUSSIONS

A microfluidic chip with an array of single cell trapping geometries [5] for cell trapping is designed and fabricated using standard soft lithography technology. 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 2 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.

The statistical results of the cell trapping is shown in Fig 2. 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. Figure 2: The statistic results of cell trapping

20 µm

20 µm0 µs 2 µs 4 µs 6 µs 8 µs 10 µs 12 µs 14 µs

0 µs 2 µs 4 µs 6 µs 8 µs 10 µs 12 µs 14 µs(a)

(b) Figure 3: 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.

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. 3(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. 3(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. 3(c). The residue of the bubble always stays at the same location after the collapse of the bubble.

The selected high-speed images of the dynamics process of the poration of single Myeloma cell are shown in Fig. 4. A

Myeloma cell is trapped first at the image t = -2 µs. 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.

0 1 20.0

0.1

0.2

0.3

0.4

0.5

Fra

ctio

n o

f Tra

ps

(N =

94)

Number of Cell Trapped

95

‐2 µs

cell

0 µs

bubble

4 µs 6 µs 10 µs 8 ms Figure 4: Single-cell membrane poration dynamics process. A Myeloma cell is trapped, and a bubble close to the cell is crated. The bubble starts to collapse and the cell is deformed and porated. The deformation of the cell is obvious in the image t = 6µs. The scale bar is 10µm.

28 s20 s12 s4 s2 s0 s

Figure 5: The Trypan blue uptake process of the porated cell The selected high speed images of the Trypan blue uptake process are shown in Fig. 4. 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.

CONCLUSIONS

In conclusion, a method for single suspension cell membrane poration is developed using cavitation bubble-induced jet flow. A microfluidic chips with an array of single cell trapping geometries is designed and fabricated using standard soft-lithography technology. Myeloma cells suspended in Trypan blue saline solution are flushed into the chip and are 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 collapses asymmetrically and induces one jet flow towards the trapped cell. The cell is deformed an porated by the jet flow and Trypan blue molecules diffusing to the cytosol of the cell indicates the poration is realized by the jet flow.

REFERENCES [1] R. E. A. Arndt, “Cavitation in Fluid Machinery and Hydraulic Structures,” Annu. Rev. Fluid. Mech., 13, 273 – 328

(1981). [2] M. S. Plesset and R. B. Chapman, “Collapse of An Initially Spherical Vapour Cavity in the Neighbourhood of A Solid

Boundary,” J. Fluid. Mech., 47, 283 – 290 (1971). [3] W. Laruterborn and H. Bolle, “Experimental Investigations of Cavitation-Bubble Collapse in Neighbourhood of A

Solid Boundary,” J. Fluid. Mech., 72, 391 – 399 (1975). [4] R. Dijkink, and C. D. Ohl, “Laser-induced cavitation based micropump,” Lab Chip, 8, 1676 – 1681 (2008). [5] Z. G. Li, K. Ando, J. Q. Yu, A. Q. Liu, J. B. Zhang and C. D. Ohl, “Fast on-demand droplet fusion using transient

cavitation bubbles,” Lab Chip, 10, 1879 – 1885 (2011). [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, 7, 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, 105, 078101 (2010). [8] L. Rayleigh, “On the pressure developed in a liquid during the collapse of a spherical cavity,” Philos. Mag., 34, 94 – 98

(1917). [9] J. R. Blake, and D. C. Gibson, “Cavitation bubbles near boundaries,” Annu. Rev. Fluid Mech., 19, 99 – 123 (1987). CONTACT *A. Q. Liu, Tel: +65-6790 4336; Fax: +65-6793 3318; Email: eaqliu@ntu.edu.sg

96