Download a single-cell membrane dynamic from poration to restoration by

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*
1
School of Electrical & Electronic Engineering
School of Chemical and Biomedical Engineering
3
School of Physical and Mathematical Sciences, Nanyang Technological University, SINGAPORE 637371
4
Data Storage Institute, 5 Engineering Drive I, SINGAPORE 117608
2
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
Jet Formation
Bubble
Bubble
Expansion
Jet Formation &
Two Vortices
Bubble
Collapse
Cell Restoring
& Trypan Blue
Upatake
Cell
(a)
(b)
(c)
(d)
(e)
(f)
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
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15th International Conference on
Miniaturized 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)).
20 µm
20 µm
0 µs
0 µs
2 µs
2 µs
4 µs
4 µs
6 µs
6 µs
8 µs
8 µs
0.5
0.4
Fraction of Traps
(N = 94)
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, SigmaAldrich) 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.
0.3
0.2
0.1
0.0
0
1
Number of Cell Trapped
2
Figure 2: The statistic results of cell trapping
10 µs
10 µs
12 µs
14 µs
12 µs
(a)
14 µs
(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.
95
bubble
cell
‐2 µs
0 µs
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.
0 s
2 s
4 s
12 s
20 s
28 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 softlithography 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
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(1981).
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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
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[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
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CONTACT
*A. Q. Liu, Tel: +65-6790 4336; Fax: +65-6793 3318; Email: [email protected]
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