A new micromachined cell lysis device is developed. It is designed for miniature bio-analysis systems where cell lysing is needed to obtain intracellular materials for further analysis such as DNA identification. It consists of muti-electrode pairs to apply electric fields to cells. We adopt the means of using electric field lysing because it can greatly simplify purification steps for preparation of biological samples, when compared to conventional chemical methods. Yeast, Chinese cabbage, radish cells and E. coli are tested with the device. The lysis of yeast, Chinese cabbage, radish cells is observed by a microscope. The experimental observation suggests E. coli are also lysed by the pulsed electric field. The range of electric field for the lysis is on the order of 1 kV/cm to 10 kV/cm. In addition, for practical reasons, we reduce the voltage required for lysing to less than 10 V by making the electrode gap on the order of microns

Lee, S. W.

Tai, Y. C.

Yowanto, H.

English

Caltech Authors - Main

A MICRO CELL LYSIS DEVICE 
S. W. Lee, H. Yowanto* and Y. C. Tai 
Caltech Micromachining Laboratory, 
Electrical Engineering, 136-93, 
California Institute of Technology, 
Pasadena, CA 91 125, USA 
*Clinical Micro Sensors, Inc., 
10 1 Waverly Drive, 
Pasadena, CA 91 105, USA 
ABSTRACT 
A new micromachined cell lysis device is developed. It 
is designed for miniature bio-analysis systems where 
cell lysing is needed to obtain intracellular materials for 
further analysis such as DNA identification. It consists 
of muti-electrode pairs to apply electric fields to cells. 
We adopt the means of using electric field lysing 
because it can greatly simplify purification steps for 
preparation of biological samples, when compared to 
conventional chemical methods. Yeast, Chinese 
cabbage, radish cells and E. coli are tested with the 
device. The lysis of yeast, Chinese cabbage, radish cells 
is observed by a microscope. The experimental 
observation suggests E. coli are also lysed by the pulsed 
electric field. The range of electric field for the lysis is 
on the order of 1 kV/ cm to I O  kVlcm. In addition, for 
practical reasons, we reduce the voltage required for 
lysing to less than 10 V by making the electrode gap on 
the order of microns. 
INTRODUCTION 
Cell lysing is defined as disrupting cells by physical, 
chemical, mechanical or enzymatic means in order to 
obtain intracellular materials. Physical means includes 
osmotic shock or pressure. Detergents, solvents and 
antibiotics are used for the chemical method. 
Mechanical shear methods are also used to disrupt cells 
[l]. The most common method used in biotechnology 
labs is the chemical method because the protocols are 
well established. Interestingly, cell lysing by pulsed 
electric fields has been reported using macro 
electroporation systems, but a micro device for doing so 
has not yet been developed [2 - 41. The applications of 
electroporation have included the introduction of both 
foreign DNA and RNA to a variety of plant, animal, 
bacterial, and yeast cells. The electric field makes 
micro-pores on the cell membrane and cells permeable 
to the medium so that DNA or RNA can be introduced 
into cells. The micro-pores are resealable in a moment. 
If the electric field is high enough, it will cause the 
irreversible breakdown of the cell membrane and will 
sometimes lyse cells. The goal of this project is the 
development of a micro cell-lysing device that will be 
used on a small number of cells. It can greatly reduce 
purification steps for preparing bio-samples in 
comparison to chemical method. It also implies the 
reduction of the number of valves and pumps when a 
micro bio-analytic system is developed. The micro cell 
lysis device can reduce the voltage required for cell 
lysis because the electrode gap can be easily fabricated 
in a size comparable to the size of biological cells. 
Theory 
The bilayer structure of a cell membrane is a dielectric. 
When a cell is exposed to an external electric field, a 
X 
Fig.1 Induction of a transmembrane potential 
transmembrane potential, AT, is induced. Fig. 1 shows 
the induction of transmembrane potential. If the 
transmembrane potential is higher than about 1 V, the 
membrane are permeable to the outside medium. For a 
spherical cell of radius a,  the transmembrane potential 
can be expressed as [3,5,6] 
where E is the applied electric field strength and the 
angle, 8, is between the field line and the normal to the 
point of interest in the membrane. The mechanism of 
443 0-7803-4412-X/98/$10.00 0 1998 IEEE 
electroporation is not filly understood. The most widely 
accepted model for electroporation, the 
electromechanical compression of the cell membrane, 
was proposed by Zimmermann [7, 81. The attraction of 
opposite charges induced on the inner and outer 
membrane generates compression pressure, which 
makes the membrane thinner. If the electric field 
strength exceed a critical value, when the 
transmembrane potential is approximately 1 V, the cell 
membrane becomes permeable to the medium. The 
poration of cell membrane can be reversible or 
irreversible depending on the electric field strength and 
duration of pulse. The irreversible breakdown of 
membrane cause cell membranes to burst open [9], or as 
the osmotic pressure of the cytosol and the external 
medium become unbalanced and the cells swell, the 
membrane is tom as a result of the over-swelling [3]. 
the fabricated and packaged device is shown in Fig. 4 
and 5, respectively. 
Oxidation 
Cr-Au evaporation and pattern 
Parylene C deposition and pattern 
Inlet Outlet 
DESIGN AND FABRICATION 
The schematic operation of the device is shown in Fig. 
2. The way of using the device is as follows. First, the 
cells and the medium are pumped into the channel. 
Next, the cells are attracted to the sharp point of the 
electrode by dielectrophoretic force using AC voltage. 
Then, they are lysed by pulsed electric fields. The edge 
of the electrodes is sharp in order to have field 
Fig. 2 Schematic view of cell lysis device 
concentration. The gap between electrodes in our device 
is 5pm. Parylene is used to make blocks between 
electrode pairs. 
The fabrication sequence of the cell lysis device is 
shown in Fig. 3. A 5000 A silicon dioxide is thermally 
grown on a silicon substrate. CrIAu (200 A I5000 A) is 
thermally evaporated and patterned for electrodes. 4 pm 
thick poly-p-xylylene (Parylene C) - is deposited and 
patterned to make blocks between electrode pairs. Then 
the device is bonded to a glass substrate with inlets, 
outlets and channel. The 30 pm high channel in the 
glass was made by timed wet etching in buffered HF. 
For attaching tubes, 750 pm inlet and outlet holes are 
made by drilling. Silastic tube whose inner diameter is 
300 pm is connected to the holes. The photograph of 
Packaging with glass channel 
Fig. 3 Fabrication steps and fully packaged device 
Fig. 4 Photograph of a fabricated device 
Fig. 5 Photograph of a fabricated device 
444 
LYSING EXPERIMENTS AND RESULTS 
The pulse generator was made with multivibrator 
circuits, a solid-state relay and a power MOSFET. A 
typical waveform is shown in Fig. 6. The pulse duration 
and timing are controlled by multivibrators (74LS 123). 
(a) Before pulsed voltage 
Fig. 6 Plot of waveform for cell lysis 
Yeast (Saccharomyces cerevisiae) protoplasts and 
Escherichia coli (strain ATCC 25922) were tested for 
lysis. Cabbage and radish protoplasts were also 
examined with a different type of electrodes. The lysis 
of yeast cells, cabbage and radish protoplasts was 
observed and determined under a microscope. In case of 
E. coli, a certain volume of the lysed cells was collected 
to test their viability after applying the lysing voltage. 
Yeast cells 
Fig. 7a and 7b show the yeast cells before and after 
applying pulsed voltages. The diameters of cells are in 
the range of 4 - 6 pm. Fig. 7a shows the when 2MHz, 
6V AC voltage is applied. The yeast cells are attracted 
to the electrodes. Fig. 7b shows that, after lysing the 
cells, they cannot be attracted to the electrodes using the 
2MHz AC voltage [9]. The lysis of yeast cells is also 
examined by the microscope as cells are collected just 
after lysis. Fig. 7c shows yeast cells that are not exposed 
to the electric fields and Fig. 7d shows the cells after 
lysing. In Fig 7d, only membrane debris is found after 
pulsed electric field treatment. The black dots in Fig. 7d 
are cell debris. Fig. 8 shows the lysing rate with 
different electric fields and pulse duration. The rate 
increases with higher lysing voltage and duration. 
However, excessive pulse voltage and duration can 
cause electrolysis which generates bubbles. The 
optimum value for yeast cell lysing was achieved at 
1 O O p  and 20V. 
E. coli 
A pure colony of E. coli was incubated overnight in 
Tryptic Soy Broth (Difco L/N 104073JB). The sizes of 
(b) After pulsed voltage 
(c) Yeast cells not treated by pulsed voltage 
(d) Collected yeast after treatment of pulse 
Fig. 7 Photograph of yeast cells before and after lysing 
445 
100 I 
e 
loops ,  5 pulse / 
60 
.- E: 4 0 -  
v) 
J 
A 30- 
20 
10 
- 
- 
0 -  
0 5 10 15 20 
Applied Voltage[V] 
Chinese cabbage 
protoplasts 
Radish 
protoplasts 
Yeast 
protoplasts 
Fig. 8 Plot of lysing rate for yeast cells 
> 590 kVlcm at 
1 MHz 
> 880 kVlcm at 
1 MHz 
Not observed up 
to 21 kV/cm at 2 
MHZ 
cells are between 2 - 4 pm. To ensure that the cells are 
lysed, the pulsed voltages were continuously applied 
while the E. coli cells were slowly injected at the inlet. 
A 3.5V 500 ps pulse was applied every two seconds. 
Higher voltage than 3.5 V caused. It is very difficult to 
visibly determine by the microscope if cells are lysed or 
not. As a result, the cells were collected and cultured on 
Tryptic Soy agar plate (Difco L/N 97898JD) ovemight 
to observe if the cells were actually lysed. Fig. 9 shows 
the photograph after the 24 hour incubation of cells. 
The cells without the pulse treatments grew in the left 
petri dish, but cells given the pulse treatments did not 
(right petri dish). This suggests that E. coli cells were 
also lysed. 
Fig. 9 Photograph of incubated E. coli cells 
Chinese cabbage and radish protoplasts 
Chinese cabbage and radish protoplasts were prepared 
by removing cell walls in an enzyme solution. The 
average diameters of cells are 30 and 35 pm, 
respectively. The cells are pipetted to a 60 pm gap 
electrodes. Lysis of cells using AC voltage was 
observed above 880 Vlcm for radish cells and 590 Vicm 
for Chinese cabbage cells at 1 MHz. The cells were 
deformed and eventually burst. A Chinese cabbage cell 
is attracted on the electrode by dielectrophoretic force 
in Fig. loa. It is lysed when the voltage is increased and 
shown in Fig. lob. The pulsed electric field alw Ivsed 
cells immediately after application. With 1 ins square 
pulse, Chinese cabbage cells are lysed above 1 .FkVIcm 
and radish cells above 1.75 kV/cm. Larger cells are 
more easily lysed in low electric fields than smaller 
cells. Table 1 shows a summary of the lysing conditions 
of our experiments. Lysis or deformation of yeast and 
E. coli were not observed until 21 kVicm at 2MHz. 
Fig. 10 Photograph of lysing Chinese cabbage cell 
Table 1. Summary of Lysis condition 
I AC 
E. coli Not observed up 
to 21 kVIcm at 2 
DISCUSSION 
DC 
(square pulse) 
> 1.5 kVlcm at 
lms 
> 1.75 ltV/cm at 
lms 
> 10 kVlcm at 
IO0 ps 
> 7 kVicm at 
500 ps 
Cell lysis by electric field can be performed with AC or 
DC electric fields. Some difference in lysis between the 
microbials on the order of few pm versus the plant cells 
on the order of 20 - 40 pm, is observed. The 
protoplasts of plant cells are stretched by AC electric 
fields; dielectrophoretic force pulls the cells between 
the electrodes. The cell membranes can be torn out if 
the strength of electric filed is high enough. For yeast 
and E. coli, cell lysis by AC electric fields, up to 20 
kVIcm, was not observed. The dielectrophoretic force 
on the cells was not high enough to breakdown the cell 
membrane because the surface to volume ratio is higher 
than in the plant cells. For lysis by DC, the plant cells 
446 
are instantly burst open and lysed by pulsed electric 
fields. Yeast cells show negative dielectrophoresis after 
being exposed to a pulsed electric field and have a 
spherical shape, but the color is slightly different. 
Normal cells are not observed after collecting the cells. 
It supports Tsong’s theory [3]: the osmotic pressures 
inside and outside the cells are unbalanced causing the 
cells to swell and eventually burst due to over-swelling 
131. 
CONCLUSION 
Cell lysing has been demonstrated with the 
micromachined cell lysis device. This device is 
promising for cell lysing and can be used for bio- 
analysis systems. The protoplasts of cells are lysed by 
AC and DC electric fields. The microbials are mostly 
lysed by pulsed electric fileds 
ACKNOWLEDGMENTS 
This work is supported by the DARPA MICRO- 
FLUMES program under Naval Ocean Systems Center 
Contract N6600 1 -96-C-83632. The authors would like 
to thank Xing Yang for help with processing. 
REFERENCES 
[I]  Biochemical Engineering and biotechnology 
handbook, Stckton press, New York, 1991, pp. 
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[2] U. Zimmermann, P. Scheurich, G. Pilwat, and R. 
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technology,” Angew. Chem. Ed. Engl., Vol. 20, pp. 
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[4] G. W. Bates, “Electrofusion of Plant Protoplast and 
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[6] P. Lindner, E. Neumann, and K. Rosenheck, 
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447 


A micro cell lysis device

A study of fabrication and applications of micromachined cell manipulating devices,

Biochemical Engineering and biotechnology handbook, Stckton press,

Cells with manipulated fuctions: New perspectives for cell biology, medicine and technology,”

Dielectric properties and ion mobility in erythrocytes,”

Electrical breakdown, electropermeabilization and electrofkion,” Rev.

Electrofusion of Plant Protoplast and the production of Somatic Hybrids,” in: Guide to Electroporation and Electrofusion

Electroporation of Cell Membranes: Mechanism and Application,” in:

Kinetics of permeability changes induced by electric impulses in chromaffin granules,”

https://authors.library.caltech.edu/12619/1/LEEmems98.pdf

A micro cell lysis device

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