PARALLEL MONITORING OF SINGLE CELL
RESPIRATION ACTIVITY BY USING
MICROARRAYED OXYGEN SENSORS
M.Suzuki, T.Yamada, S.Kato and Y.Iribe
University of Toyama, JAPAN
ABSTRACT
This paper reports a parallel monitoring method for single cell respiration activity by using a microarrayed oxygen sensor. Optical oxygen sensor film was prepared
by coating the mixture of 0.5g/l dichlorotris (1,10-phenanthroline) ruthenium (II)
and 2.5% Nafion onto a diamond like carbon sputtered slide glass. Micro well (10
μm i.d.) array was prepared with carbon black doped epoxy resin onto the sensor
film. The prepared microarrayed oxygen sensor enabled independent oxygen sensing
in each micro well, and the difference of respiration activity of single cell (mouse
lymphocyte) could be detected in a microarrayed oxygen sensor.
KEYWORDS: Single cell, Respiration activity, Oxygen sensor, Sensor array
INTRODUCTION
We have developed microarrayed chemical sensors for parallel monitoring of
single cell activity [1]. These sensors consist of optical sensor film for pH or oxygen,
and micro well array prepared with PDMS. Since PDMS sheet is transparent and
oxygen permeable, the change of background level was serious problem for this sensor array. In this paper, micro well array was prepared with carbon black doped epoxy resin onto the sensor film. The prepared microarrayed oxygen sensor enabled
independent oxygen sensing in each micro well, and the difference of respiration activity of single cell could be detected in a microarrayed oxygen sensor.
EXPERIMENTAL
The structure of microarrayed oxygen sensors was shown in Figure 1. Oxygen
sensor film was prepared by coating the mixture of 0.5g/l dichlorotris (1,10phenanthroline)ruthenium (II) and 2.5% Nafion onto a DLC(diamond like carbon)
sputtered slide glass (Gene slide, Toyo Kohan Co.Ltd.).
Micro well array prepared with carbon black
Micro
wellepoxy
array prepared
doped
resinwith carbon black doped epoxy resin
DLC sputtered
glassglass
DLC
sputtered
Oxygen
sensor
(ruthenium
Oxygen sensor
filmfilm
(ruthenium
complex) complex)
Figure 1. Structure of microarrayed oxygen sensors
Micro well array (well diameter 10 μm) was prepared onto the sensor film with
the mixture of epoxy resin and carbon black powder (24nm i.d.) in order to intercept
Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences
October 12 - 16, 2008, San Diego, California, USA
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oxygen and light. Fluorescence intensity of each well was measured by using a laser
conforcal high resolution microarray scanner (Ex. 473nm, Em. 583nm , CRBIOIIeFITC, Hitachi Soft Corp.). Mouse lymphocytes obtained from Slc:ICR mouse spleen
were used throughout this study as a cell sample.
1.15
4000
3500
3000
2500
2000
1500
1000
500
0
1.1
I / Io [-]
⊿Intensity [a.u.]
RESULTS AND DISCUSSION
Micro well array sheet prepared with carbon black doped epoxy resin did not
permeate oxygen and light. Background fluorescence was negligible and was not affected by the fluorescence change in micro wells (Figure 2). Therefore independent
oxygen sensing in each micro well became possible.
Then, relationship between fluorescence change and the difference of respiration
activity of cells was evaluated. The mixture of viable cells and dead cells were filled
with 100 μm i.d. micro wells and fluorescence intensity change was recorded. After
the fluorescence measurement, cells were stained with calcein-AM (green, viable
cells) and propidium iodide (PI)(red, dead cells), and ratio of viable cells was calculated. As shown in Figure 3, good correlation was observed between the ratio of viable cells and fluorescence intensity increase. This result shows the difference of
respiration activity can be detected by fluorescence intensity change of ruthenium
complex.
1.05
1
0.95
0.9
0.85
wells
0.8
background
0
10 20 30 40 50 60 70 80 90 100
Ratio of ｌiving cells [%]
Figure 2. Effect of fluorescence change in
micro wells on background fluorescence
Fluorescence change between 5%Na2SO3
and distilled water was compared between
carbon black doped epoxy resin well
sheet (occupied columns) and conventionnal PDMS well sheet (open columns).
Figure 3. Ratio of living cells and
fluorescence change in 100μm i.d.
oxygen sensing wells
I0:Fluorescence intensity before cell
injection, I:Fluorescence intensity
after 20min incubation with cells
Total cell conc.: 2x107 cells/ml
Single viable or dead cell was filled in 10 μm i.d. micro wells and the difference
of respiration activity was detected by the difference of fluorescence intensity. Figure 4 shows an optical micrograph ((a)) and pseudo-colored fluorescence images obtained by a microarray scanner ((b),(c)) of the micro wells filled with cells. As
shown in Figures 5, fluorescence intensity was increased in the wells filled with viable cells. This shows large oxygen consumption by respiration. Contrarily fluorescence intensity change was very small in the wells filled with dead cells or no cells.
These results show the present sensor array can detect the difference of respiration
activity on single cell level.
Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences
October 12 - 16, 2008, San Diego, California, USA
439
1
2
3
4
5
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7
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(a)Micrograph
(b)Before incubation
(c)After 30min
Figure 4. Micro wells filled with viable and dead cells analyzed in Figure 5.
Wells filled with a viable cell was indicated with a square, and wells filled with a
dead cell was indicated with a circle.
1.8
I / Io [-]
1.6
1.4
1.2
1
0.8
死細胞平均
生細胞平均
No細胞なし
cells Dead
cells Viable
cells
(n=16)
(n=6)
(n=3)
Figure 5. Comparison of fluorescence intensity change in micro wells
filled with a viable or dead cell (Incubation tim: 30 min.)
ACKNOLEDGMENTS
This work was supported by a Grant-in-Aid for Scientific Research on Priority
Areas (No.455) (No.18048018).
REFERENCES
[1] M.Suzuki, H.Nakabayashi, Y.Jing, M.Honda, Optical pH and oxygen sensing
for micro-arrayed cell chips, Micro Total Analysis Systems 2005, Vol.2, 14821484 (2005).
Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences
October 12 - 16, 2008, San Diego, California, USA
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